Structural carotenoid analogs for the inhibition and amelioration of disease

ABSTRACT

A method for inhibiting and/or ameliorating the occurrence of diseases associated with reactive oxygen species, reactive nitrogen species, radicals and/or non-radicals in a subject whereby a subject is administered a carotenoid structural analog, either alone or in combination with another carotenoid analog, or co-antioxidant formulation. The analog or analog combination is administered such that the subject&#39;s risk of experiencing diseases associated with reactive oxygen species, reactive nitrogen species, radicals and/or non-radicals may be thereby reduced. The analog or analog combination may be administered to a subject for the inhibition and/or amelioration of ischemia-reperfusion injury. The analog or analog combination may be administered to a subject for the inhibition and/or amelioration of liver disease. The analog or analog combination may be administered to a subject for the inhibition and/or amelioration of cancer. The analog or analog combination may be administered to a subject for the inhibition and/or amelioration of cardiac arrhythmia and/or sudden cardiac death. The analog or analog combination may be administered to a subject for the inhibition and/or amelioration of any disease that involves production of reactive oxygen species, reactive nitrogen species, radicals and/or non-radicals. In one embodiment, a water-soluble and/or water-dispersible astaxanthin analog is particularly effective. This invention further includes pharmaceutical compositions comprising structural carotenoid analogs either alone or in combination.

PRIORITY CLAIM

This application is a Continuation of patent application Ser. No.10/629,538 entitled “Structural Carotenoid Analogs for the Inhibitionand Amelioration of Disease” filed on Jul. 29, 2003, now U.S. Pat. No.7,145,025, which claims priority to Provisional Patent Application No.60/399,194 entitled “Structural Carotenoid Analogs for the Inhibitionand Amelioration of Reperfusion Injury” filed on Jul. 29, 2002;Provisional Patent Application No. 60/467,973 entitled “StructuralCarotenoid Analogs for the Inhibition and Amelioration of Disease” filedon May 5, 2003; Provisional Patent Application No. 60/472,831 entitled“Structural Carotenoid Analogs for the Inhibition and Amelioration ofDisease” filed on May 22, 2003; Provisional Patent Application No.60/473,741 entitled “Structural Carotenoid Analogs for the Inhibitionand Amelioration of Disease” filed on May 28, 2003; and ProvisionalPatent Application No. 60/485,304 entitled “Structural CarotenoidAnalogs for the Inhibition and Amelioration of Disease” filed on Jul. 3,2003, all of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1 . Field of the Invention

The invention generally relates to the fields of medicinal and syntheticchemistry. More specifically, the invention relates to the synthesis anduse of carotenoid analogs.

2. Description of the Relevant Art

Cardiovascular disease (CVD), and specifically coronary artery disease(CAD), remains the leading cause of death in the United States andworldwide. CVD is a leading cause of mortality and morbidity in theworld. Small to moderate reductions in cardiovascular risk, which leadto decreased emergency department visits and hospitalizations for acutecoronary syndromes, can yield substantial clinical and public healthbenefits.

Extensive research with antioxidants has shown that they are effectivetherapeutic agents in the primary and secondary prevention ofcardiovascular disease. CVD remains the leading cause of death for allraces in the U.S.; now, approximately 60 million Americans have someform of CVD. Life expectancy in the U.S. would increase by almost 7years if CVD could be eliminated. The absolute number of deaths due toCVD has fallen since 1996; however, it remains the single largest causeof death in the United States, with a total annual healthcare burden ofgreater than $300 billion (including heart attack and stroke).

Ischemia is the lack of an adequate oxygenated blood supply to aparticular tissue. Ischemia underlies many acute and chronic diseasestates including, but not limited to:

-   -   Myocardial infarction, or MI    -   Unstable angina    -   Stable angina pectoris    -   Abrupt reclosure following percutaneous transluminal coronary        angioplasty (PTCA)    -   Thrombotic stroke (85% of the total number of strokes)    -   Embolic vascular occlusion    -   Peripheral vascular insufficiency    -   Organ transplantation    -   Deep venous thrombosis, or DVT    -   Indwelling catheter occlusion        Ischemia may also become a problem in elective procedures such        as: scheduled organ transplantation; scheduled coronary artery        bypass graft surgery (CABG); and scheduled percutaneous        trausluminal coronary angioplasty (PTCA). Common to each of        these settings is the phenomenon of reperfusion injury: the        production of reactive oxygen species (ROS) upon reintroduction        of oxygenated blood flow to a previously ischemic area, with        subsequent paradoxical additional tissue damage. In particular,        the use(s) of thrombolytic therapy in acute myocardial        infarction (AMI) and acute thrombotic stroke—as well as surgical        revascularization with PTCA—are typically associated with the        reperfusion of ischemic myocardium and/or brain. Clinical        outcome is improved with the achievement of early patency after        acute thrombosis, however, not without cost (i.e., “reperfusion        injury”).

Current therapy allows for reperfusion with pharmacologic agents,including recombinant tissue-type plasminogen activator (r-TPA),Anistreplase (APSAC), streptokinase, and urokinase. Recent studies haveshown the best clinical outcome after AMI occurs with early surgicalreperfusion. However, surgical reperfusion is available at only 15 to 20percent of care centers in the United States, and much fewer worldwide.It is likely, therefore, that pharmacologic reperfusion will remainclinically relevant and important for the foreseeable future.Thrombolytic therapy is unsuccessful in reperfusion of about 20% ofinfarcted arteries. Of the arteries that are successfully reperfused,approximately 15% abruptly reclose (within 24 hours). Measures ofsystemic inflammation (e.g., serum levels of C-reactive protein or CRP)correlate strongly with clinical reclosure in these patients. Myocardialsalvage appears to be maximal in a 2 to 6 hour “therapeutic window”subsequent to acute plaque rupture and thrombosis. In acute thromboticor thromboembolic stroke, this therapeutic window is even narrower,generally less than 3 hours post-thrombosis. Recombinant tissue-typeplasminogen activator administered within 3 hours of ischemic strokesignificantly improves clinical outcome, but increases the risk ofhemorrhage.

During a period of ischemia, many cells undergo the biochemical andpathological changes associated with anoxia but remain potentiallyviable. These potentially viable cells are therefore the “battleground”in the reperfusion period. Ischemia creates changes in the affectedtissue, with the potential final result of contraction band and/orcoagulation necrosis of at-risk myocardium. Pathologic changes inischemic myocardium include, but are not limited to:

-   -   Free radical and ROS production    -   ATP loss and defective ATP resynthesis    -   Creatine phosphate loss    -   Extracellular potassium loss    -   Active tension-generating capacity loss of myocardium    -   Cellular swelling    -   Acidosis    -   Loss of ionic homeostasis    -   Structural disorganization    -   Electrical instability and arrhythmogenesis    -   Lipid membrane peroxidation    -   Glutathione and other endogenous/exogenous antioxidant depletion        (including vitamins C and E and carotenoids)        Rescue of ischemic myocardium that has not irreversibly reached        the threshold of necrosis is the focus of intervention in        reperfusion injury.

Gap junctions are a unique type of intercellular junction found in mostanimal cell types. They form aqueous channels that interconnect thecytoplasms of adjacent cells and enable the direct intercellularexchange of small (less than approximately 1 kiloDalton) cytoplasmiccomponents. Gap junctions are created across the interveningextracellular space by the docking of two hemichannels (“connexons”)contributed by each adjacent cell. Each hemichannel of is an oligomer ofsix connexin molecules.

Connexin 43 was the second connexin gene discovered and it encodes oneof the most widely expressed connexins in established cell lines andtissues. Gap junctions formed by connexin 43 have been implicated indevelopment, cardiac function, and growth control.

One common manifestation of CVD is cardiac arrhythmia. Cardiacarrhythmia is generally considered a disturbance of the electricalactivity of the heart that manifests as an abnormality in heart rate orheart rhythm. Patients with a cardiac arrhythmia may experience a widevariety of symptoms ranging from palpitations to fainting (“syncope”).

The major connexin in the cardiovascular system is connexin 43. Gapjunctional coordination of cellular responses among cells of thevascular wall, in particular the endothelial cells, is thought to becritical for the local modulation of vasomotor tone and for themaintenance of circulatory homeostasis. Controlling the upregulation ofconnexin 43 may also assist in the maintenance of electrical stabilityin cardiac tissue. Maintaining electrical stability in cardiac tissuemay benefit the health of hundreds of thousands of people a year withsome types of cardiovascular disease [e.g., ischemic heart disease (IHD)and arrhythmia], and may prevent the occurrence of sudden cardiac deathin patients at high risk for arrhythmia.

Cancer is generally considered to be characterized by the uncontrolled,abnormal growth of cells. Connexin 43, as previously mentioned, is alsoassociated with cellular growth control. Growth control by connexin 43is likely due to connexin 43's association with gap junctionalcommunication. Maintenance, restoration, or increases of functional gapjunctional communication inhibits the proliferation of transformedcells. Therefore, upregulation and/or control of the availability ofconnexin 43 may potentially inhibit and/or ameliorate the spread ofcancerous cells.

Chronic liver injury, regardless of etiology, may lead to a progressivespectrum of pathology from acute and chronic inflammation, to earlystage fibrosis, and finally to cirrhosis and end-stage liver disease(ESRD). A cascade of inflammatory events secondary to the initiatinginjury, including the release of cytokines and the formation of reactiveoxygen species (ROS), activates hepatic stellate cells (HSC). HSCproduce extracellular matrix components (ECM), including collagen, andare critical in the process which generates hepatic fibrosis.

End-stage liver disease [manifested as either cirrhosis orhepatocellular carcinoma (HCC)] is the eighth leading cause ofdisease-related death in the United States. Chronic inflammation in theliver resulting from viral infection, alcohol abuse, drug-inducedtoxicity, iron and copper overload, and many other factors can initiatehepatic fibrosis. By-products of hepatocellular damage activate Kupffercells, which then release a number of cytokines, ROS(including inparticular superoxide anion), and other paracrine and autocrine factorswhich in turn act upon hepatic stellate cells (HSC). It is now believedthat the lynchpin cell in the fibrogenetic cascade is the HSC, the celltype responsible for the production of ECM. In vitro evidencedemonstrates that ROS can induce HSC cells. Elevated levels of indirectmarkers of oxidative stress (e.g., thiobarbituric acid reactive speciesor TBARS) are observed in all patients with chronic liver disease. Inaddition, levels of gluthathione, glutathione peroxidase, superoxidedismutase, carotenoids, and α-tocopherol (vitamin E) are significantlylower in patients with chronic liver disease. Supplying these endogenousand/or exogenous antioxidants reverses many of the signs of chronicliver disease, including both surrogate markers for the disease process,as well as direct measurements of hepatic fibrosis. Therefore, they arelikely potent agents for therapeutic intervention in liver disease.

SUMMARY

In some embodiments, the administration of structural analogs ofcarotenoids may inhibit and/or ameliorate the occurrence of diseases insubjects. Maladies which may be treated with structural analogs ofcarotenoids may include any disease that involves production of reactiveoxygen species and/or other radical species (for example singlet oxygen,a reactive oxygen species but not a radical). In some embodiments,water-soluble analogues of carotenoids may be used to treat a diseasethat involves production of reactive oxygen species. Oxidation of DNA,proteins, and lipids by reactive oxygen species and other radical andnon-radical species has been implicated in a host of human diseases.Radicals may be the primary cause for the following conditions, may makethe body more susceptible to other disease-initiating factors, mayinhibit endogenous defenses and repair processes, and/or may enhance theprogression of incipient disease(s). The administration of structuralanalogs of carotenoids by one skilled in the art—including considerationof the pharmacokinetics and pharmacodynamics of therapeutic drugdelivery—is expected to inhibit and/or ameliorate said diseaseconditions. In the first category are those disease conditions in whicha single organ is primary affected, and for which evidence exists thatradicals and/or non-radicals are involved in the pathology of thedisease. These examples are not to be seen as limiting, and additionaldisease conditions will be obvious to those skilled in the art.

-   -   Head, Eyes, Ears, Nose, and Throat: age-related macular        degeneration (ARMD), retinal detachment, hypertensive retinal        disease, uveitis, choroiditis, vitreitis, ocular hemorrhage,        degenerative retinal damage, cataractogenesis and cataracts,        retinopathy of prematurity, Meuniere's disease, drug-induced        ototoxicity (including aminoglycoside and furosemide toxicity),        infectious and idiopathic otitis, otitis media, infectious and        allergic sinusitis, head and neck cancer;    -   Central Nervous System (brain and spinal cord): senile dementia        (including Alzheimer's dementia), Neuman-Pick's disease,        neurotoxin reactions, hyperbaric oxygen effects, Parkinson's        disease, cerebral and spinal cord trauma, hypertensive        cerebrovascular injury, stroke (thromboembolic, thrombotic, and        hemorrhagic), infectious encephalitis and meningitis, allergic        encephalomyelitis and other demyelinating diseases, amyotrophic        lateral sclerosis (ALS), multiple sclerosis, neuronal ceroid        lipofuscinoses, ataxia-telangiectasia syndrome, aluminum, iron,        and other heavy metal(s) overload, primary brain        carcinoma/malignancy and brain metastases;    -   Cardiovascular: arteriosclerosis, atherosclerosis, peripheral        vascular disease, myocardial infarction, chronic stable angina,        unstable angina, idiopathic surgical injury (during CABG, PTCA),        inflammatory heart disease [as measured and influenced by        C-reactive protein (CRP) and myeloperoxidase (MPO)], low-density        lipoprotein oxidation (ox-LDL), cardiomyopathies, cardiac        arrhythmia (ischemic and post-myocardial infarction induced),        congestive heart failure (CHF), drug toxicity (including        adriamycin and doxorubicin), Keshan disease (selenium        deficiency), trypanosomiasis, alcohol cardiomyopathy, venous        stasis and injury (including deep venous thrombosis or DVT),        thrombophlebitis;    -   Pulmonary: asthma, reactive airways disease, chronic obstructive        pulmonary disease (COPD or emphysema), hyperoxia, hyperbaric        oxygen effects, cigarette smoke inhalation effects,        environmental oxidant pollutant effects, acute respiratory        distress syndrome (ARDS), bronchopulmonary dysplasia, mineral        dust pneumoconiosis, adriamycin toxicity, bleomycin toxicity,        paraquat and other pesticide toxicities, chemical pneumonitis,        idiopathic pulmonary interstitial fibrosis, infectious pneumonia        (including fungal), sarcoidosis, asbestosis, lung cancer (small-        and large-cell), anthrax infection, anthrax toxin exposure;    -   Renal: hypertensive renal disease, end-stage renal disease,        diabetic renal disease, infectious glomerulonephritis, nephrotic        syndrome, allergic glomerulonephritis, type I-IV        hypersensitivity reactions, renal allograft rejection, nephritic        antiglomerular basement membrane disease, heavy metal        nephrotoxicity, drug-induced (including aminoglycoside,        furosemide, and non-steroidal anti-inflammatory) nephrotoxicity,        rhabdomyolisis, renal carcinoma;    -   Hepatic: carbon tetrachloride liver injury, endotoxin and        lipopolysaccharide liver injury, chronic viral infection        (including Hepatitis infection), infectious hepatitis (non-viral        etiology), hemachromatosis, Wilson's disease, acetaminophen        overdose, congestive heart failure with hepatic congestion,        cirrhosis (including alcoholic, viral, and idiopathic        etiologies), hepatocellular carcinoma, hepatic metastases;    -   Gastrointestinal: inflammatory bowel disease (including Crohn's        disease, ulcerative colitis, and irritable bowel syndrome),        colon carcinoma, polyposis, infectious diverticulitis, toxic        megacolon, gastritis (including Helicobacter pylori infection),        gastric carcinoma, esophagitis (including Barrett's esophagus),        gastro-esophageal reflux disease (GERD), Whipple's disease,        gallstone disease, pancreatitis, abetalipoproteinemia,        infectious gastroenteritis, dysentery, nonsteroidal        anti-inflammatory drug-induced toxicity;    -   Hematopoietic/Hematologic: Pb (lead) poisoning, drug-induced        bone marrow suppression, protoporphyrin photo-oxidation,        lymphoma, leukemia, porphyria(s), parasitic infection (including        malaria), sickle cell anemia, thallasemia, favism, pernicious        anemia, Fanconi's anemia, post-infectious anemia, idiopathic        thrombocytopenic purpura, autoimmune deficiency syndrome (AIDS);    -   Genitourinary: infectious prostatitis, prostate carcinoma,        benign prostatic hypertrophy (BPH), urethritis, orchitis,        testicular torsion, cervicitis, cervical carcinoma, ovarian        carcinoma, uterine carcinoma, vaginitis, vaginismus;    -   Musculoskeletal: osteoarthritis, rheumatoid arthritis,        tendonitis, muscular dystrophy, degenerative disc disease,        degenerative joint disease, exercise-induced skeletal muscle        injury, carpal tunnel syndrome, Guillan-Barre syndrome, Paget's        disease of bone, ankylosing spondilitis, heterotopic bone        formation; and    -   Integumentary: solar radiation injury (including sunburn),        thermal injury, chemical and contact dermatitis (including Rhus        dermatitis), psoriasis, Bloom syndrome, leukoplakia        (particularly oral), infectious dermatitis, Kaposi's sarcoma.

In the second category are multiple-organ conditions whose pathology hasbeen linked convincingly in some way to radical and non-radical injury:aging, including age-related immune deficiency and premature agingdisorders, cancer, cardiovascular disease, cerebrovascular disease,radiation injury, alcohol-mediated damage (includingWernicke-Korsakoff's syndrome), ischemia-reperfusion damage,inflammatory and auto-immune disease, drug toxicity, amyloid disease,overload syndromes (iron, copper, etc.), multi-system organ failure, andendotoxemia/sepsis.

Maladies, which may be treated with structural carotenoid analogs, mayinclude, but are not limited to, cardiovascular inflammation, hepatitisC infection, cancer (hepatocellular carcinoma and prostate), maculardegeneration, rheumatoid arthritis, stroke, Alzheimer's disease, and/orosteoarthritis. In an embodiment, the administration of water solubleanalogs of carotenoids to a subject may inhibit and/or ameliorate theoccurrence of reperfusion injury in subjects. In some embodiments, watersoluble and other structural carotenoid analogs may be administered to asubject alone or in combination with other structural carotenoidanalogs. The occurrence of reperfusion injury in a human subject that isexperiencing, or has experienced, or is predisposed to experiencemyocardial infarction, stroke, peripheral vascular disease, venous orarterial occlusion, organ transplantation, coronary artery bypass graftsurgery, percutaneous transluminal coronary angioplasty, andcardiovascular arrest and/or death may be inhibited or ameliorated bythe administration of therapeutic amounts of water soluble and/or otherstructural carotenoid analogs to the subject.

“Water soluble” structural carotenoid analogs are those analogs whichmay be formulated in aqueous solution, either alone or with excipients.Water soluble carotenoid analogs may include those compounds andsynthetic derivatives which form molecular self-assemblies, and may bemore properly termed “water dispersible” carotenoid analogs. Watersoluble and/or “water-dispersible” carotenoid analogs may be thepreferred embodiment(s) in some aspects of the current invention.

In an embodiment, the administration of water soluble analogs ofcarotenoids to a subject may inhibit and/or ameliorate some types ofcardiovascular disease associated with cardiac arrhythmia. In someembodiments, water soluble analogs of carotenoids may be administered toa subject alone or in combination with other carotenoid analogs.Carotenoid analogs may assist in the maintenance of electrical stabilityin cardiac tissue. Assistance in the maintenance of electrical stabilityin cardiac tissue may inhibit and/or ameliorate some types ofcardiovascular disease, including in particular sudden cardiac deathattributable to lethal cardiac arrhythmia.

In an embodiment, the administration of water soluble analogs ofcarotenoids to a subject may inhibit and/or ameliorate the occurrence ofliver disease in the subject. In some embodiments, water soluble analogsof carotenoids may be administered to a subject alone or in combinationwith other carotenoid analogs. The liver disease may be a chronic liverdisease such as, for example, Hepatitis C infection.

In an embodiment, the administration of water soluble analogs ofcarotenoids to a subject may inhibit and/or ameliorate the proliferationand propagation of initiated, transformed and/or cancerous cell(s). Insome embodiments, water soluble analogs of carotenoids may beadministered to a subject alone or in combination with other carotenoidanalogs. Carotenoid analogs may inhibit the proliferation rate ofcarcinogen-initiated cells. Carotenoid analogs may increase connexin 43expression. Increase of connexin 43 expression may increase, maintain,or restore gap junctional intercellular communication and thus inhibitthe growth of carcinogen-initiated cells.

Embodiments may be further directed to pharmaceutical compositionscomprising combinations of structural carotenoid analogs to saidsubjects. The composition of an injectable structural carotenoid analogof astaxanthin may be particularly useful in the therapeutic methodsdescribed herein. In yet a further embodiment, an injectable astaxanthinstructural analog is administered with another astaxanthin structuralanaolgs and/or other carotenoid structural analogs, or in formulationwith other antioxidants and/or excipients that further the intendedpurpose. In some embodiments, one or more of the astaxanthin structuralanalogs are water soluble.

In an embodiment, a chemical compound including a carotenoid may havethe general structure (I):

Each R³ may be independently hydrogen or methyl. R¹ and R² may beindependently H, an acyclic alkene with one or more substituents, or acyclic ring including one or more substituents. In some embodiments,substituents may be at least partially hydrophilic. These carotenoidderivatives may be used in a pharmaceutical composition. In oneembodiment, a pharmaceutical composition that includes carotenoidstructural analogues having general structure (I) may be used fortreating reperfusion injury.

As used herein, the terms “disodium salt disuccinate astaxanthinderivative”, “dAST”, “Cardax”, “Cardax™”, “rac”, and “astaxanthindisuccinate derivative (ADD)” represent varying nomenclature for the useof the disodium salt disuccinate astaxanthin derivative in variousstereoisomer and aqueous formulations, and represent presently preferredbut nonetheless illustrative embodiments for the intended use of thisstructural carotenoid analog. The diacid disuccinate astaxanthinderivative (astaCOOH) is the protonated form of the derivative utilizedfor flash photolysis studies for direct comparison with non-esterified,“racemic” (i.e., mixture of stereoisomers) astaxanthin. “Cardax-C” isthe disodium salt disuccinate di-vitamin C derivative (derivative XXII)utilized in superoxide anion scavenging experiments assayed by electronparamagnetic resonance (EPR) imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description as well as further objects, features andadvantages of the methods and apparatus of the present invention will bemore fully appreciated by reference to the following detaileddescription of presently preferred but nonetheless illustrativeembodiments in accordance with the present invention when taken inconjunction with the accompanying drawings.

FIG. 1 is a graphic representation of several “parent” carotenoidstructures as found in nature;

FIG. 2 depicts an effect of disodium salt disuccinate astaxanthinderivative on the reactive oxygen species superoxide anion as monitoredusing electron paramagnetic resonance (EPR) imaging;

FIG. 3 depicts an effect of a disodium salt disuccinate astaxanthinderivative/free vitamin C solution on the reactive oxygen speciessuperoxide anion as monitored using electron paramagnetic resonance(EPR) imaging;

FIG. 4 depicts a graphical representation of a relative reduction ofinfarct size in male Sprague-Dawley rats with pre-treatment using adisodium salt disuccinate astaxanthin derivative intravenous formulation(Cardax™);

FIG. 5 depicts the chemical structure of the all-trans (all-E) disodiumsalt disuccinate ester derivative of meso-astaxanthin (3R,3′S- or3S,3′R-dihydroxy-β,β-carotene4,4′-dione; dAST) synthesized for thecurrent study (shown as the all-E dianionic bolamphiphile);

FIG. 6 depicts the ultraviolet-visible absorption spectrum of dAST inethanol at 25° C. (cell length 1 cm, c=1.05×10⁻⁵M). Molar absorptioncoefficients are shown in parentheses. The second derivative curve ofthe absorption spectrum indicates the exact position of peaks in thenear-UV region and the hidden vibrational fine structure of the mainband;

FIG. 7 depicts the absorption spectrum of dAST in Ringer buffer (pH 7.4,cell length 1 cm, c=1.85×10⁻⁵ M, t=37° C.). Molar absorptioncoefficients are indicated;

FIG. 8 depicts the induced CD and UV/Vis spectra obtained by titrationof human serum albumin (HSA) with dAST in Ringer buffer solution (pH7.4) at low L/P ratios. Concentration of HSA was 1.6×10⁻⁴M and theligand was added as aliquots of DMSO stock solution (cell length 1 cm,t=37° C.). Curves measured at different L/P values are shown. Insets:molar circular dichroic absorption coefficients (Δε in M⁻¹cm⁻¹) andmolar absorption coefficients (ε in M⁻¹cm⁻¹) of the induced CD andabsorption bands calculated on the basis of total meso-carotenoidconcentration in the solution;

FIG. 9 depicts the induced CD and UV/Vis spectra obtained by titrationof HSA with dAST in Ringer buffer solution (pH 7.4) above L/P ratioof 1. Concentration of HSA was 2.3×10⁻⁴M and the ligand was added asaliquots of DMSO stock solution (cell length 1 cm, t=37 ° C.). Curvesmeasured at L/P values of 1.2, 2.0, 2.9, 4.1, 5.7 and 7.4 are shown. CDintensities increase in parallel with the ligand concentration;

FIG. 10 depicts the induced CD and UV/Vis spectra obtained by titrationof HSA with dAST in 0.1 M pH 7.4 phosphate buffer solution above L/Pratio of 1. Concentration of HSA was 2.2×10⁻⁴M and the ligand was addedas aliquots of DMSO stock solution (cell length 1 cm, t=37° C.). Curvesmeasured at L/P values of 1.2, 2.0, 2.9, 4.1, 5.7, 9.0, 10.6 and 13.1are shown. CD intensities increase in parallel with the ligandconcentration;

FIG. 11A-C depicts illustration of right-handed chiral arrangements oftwo meso-carotenoid molecules for which excitonic interactions producelong-wavelength positive and short-wavelength negative Cotton effects inthe CD spectrum. Gray-colored molecules lie behind of the plane of thepaper;

FIG. 12 depicts (upper figure): fluorescence quenching of HSA by DASTmeasured in 0.1 M pH 7.4 phosphate buffer solution at 37° C. Initial andfinal concentrations of HSA and the ligand were varied between4.2×10⁻⁶M-4.0×10⁻⁶M and 1.3×10⁻⁶M-1.4×10⁻⁵M, respectively. L/P ratiosare noted on curves. (Lower figure): effect of DMSO alone on theintrinsic flurescence of HSA. Experimental conditions are as in text;

FIG. 13 depicts the X-ray crystallographic structure of fatty acid-freeHSA. Subdomains and the two primary drug-binding sites of HSA areindicated. Dotted bar represents spatial dimension of the interdomaincleft, and asterisk indicates the position of Trp214. The inter-atomicdistance between the 3 and 3′ chiral carbon atoms of the DAST moleculeis 28 Å;

FIG. 14A-F depicts that the statistical mixture of stereoisomers of thedisodium salt disuccinate astaxanthin derivative (“rac” in FigureLegends) induces functional gap junctional communication in murineembryonic fibroblast (10T1/2) cells. Confluent cultures were treated for4 days as described in text, then assayed for the ability to transferthe fluorescent dye Lucifer Yellow. Arrows indicate the cell injectedwith Lucifer Yellow;

FIG. 15A depicts connexin 43 protein expression in cells treated withthe mixture of stereoisomers of the disodium salt disuccinateastaxanthin derivatives as assessed by quantitative Western blotanalysis. The upper bands are believed to represent the phosphorylatedforms of the protein assembled into gap junctions; lower bandsunassembled proteins (Saez, 1998). Lane 1: 1:2 ethanol (EtOH)/H₂O(solvent only negative control); Lane 2: TTNPB, a synthetic retinoid, inacetone at 10⁻⁸M (positive control); Lane 3: Retinyl acetate in acetoneat 10⁻⁵M (positive control); Lane 4: Statistical mixture (“rac”) ofstereoisomers of the disodium salt disuccinate astaxanthin derivative at10⁻⁵M delivered in a 1:2 formulation of EtOH/H₂O; Lane 5: 3R,3′Rdisodiumsalt disuccinate astaxanthin derivative at 10⁻⁵ M delivered in a 1:2formulation of EtOH/H₂O; Lane 6: 3S,3′S disodium salt disuccinateastaxanthin derivative at 10⁻⁵M delivered in a 1:2 formulation ofEtOH/H₂O; and Lane 7: Meso disodium salt disuccinate astaxanthinderivative at 10⁻⁵M delivered in a 1:2 formulation of EtOH/H₂O;

FIG. 15B depicts an immunoblot stained with Coomassie blue todemonstrate equal protein loading of all the bands. This confirms thatdifferences in immunolabeling are not an artifact due to variability intotal protein loaded and/or transferred to the membrane;

FIG. 15C depicts digital analysis of relative induction levels ofconnexin 43 protein expression by the disodium salt disuccinateastaxanthin derivative(s) versus positive and solvent-only treatedcontrols. Lanes as in FIG. 15A. The fold induction is normalized tocontrol levels of Cx43 expression in the 1:2 EtOH/H₂O treated negativecontrols set to an arbitrary unit=1.0;

FIG. 15D depicts the dose-response curve of Cx43 protein expression inmurine embryonic fibroblast cells (10T1/2) treated with the statisticalmixture of stereoisomers of the disodium salt disuccinate astaxanthinderivatives as assessed by quantitative Western blot analysis. The upperbands are believed to represent the phosphorylated forms of the proteinassembled into gap junctions; lower bands unassembled proteins. Lane 1:1:2 EtOH/H₂O (solvent only negative control). Lane 2: TTNPB in acetoneat 10⁻⁸M (positive control). Lane 3: disodium salt disuccinateastaxanthin derivative (“rac”) at 10⁻⁵M delivered in a 1:2 formulationof EtOH/H₂O. Lane 4: disodium salt disuccinate astaxanthin derivative(“rac”) at 5×10⁻⁶M delivered in a 1:2 formulation of EtOH/H₂O. Lane 5:disodium salt disuccinate astaxanthin derivative (“rac”) at 10⁻⁶Mdelivered in a 1:2 formulation of EtOH/H₂O;

FIG. 15E depicts digital analysis of relative induction levels ofconnexin 43 protein expression by the statistical mixture ofstereoisomers of the disodium salt disuccinate astaxanthin derivativeversus positive and solvent-only treated controls. Lanes as in FIG. 15D.The fold induction is normalized to control levels of Cx43 expression inthe 1:2 EtOH/H₂O treated controls set to an arbitrary unit=1.0;

FIG. 16 depicts that the statistical mixture of stereoisomers of thedisodium salt disuccinate astaxanthin derivative increases the assemblyof Cx43 immunoreactive junctional plaques. Confluent cultures of 10T1/2cells were treated for 4 days as described above with the statisticalmixture of stereoisomers of the disodium salt disuccinate astaxanthinderivative: (1) at 10⁻⁵M in 1:2 EtOH/H₂O; (2) with 1:2 EtOH/H₂O assolvent only negative control; or (3) TTNPB at 10⁻⁸M in tetralydrofuran(THF) solvent as positive control. Cells were immunostained with a Cx43antibody as described in text. Panel A: the statistical mixture ofstereoisomers of the disodium salt disuccinate astaxanthin derivative at10⁻⁵M in 1:2 EtOH/H₂O; Panel C: 1:2 EtOH/H₂O as solvent control; PanelE: TTNPB at 10⁻⁸M in tetrahydrofuran (THF) solvent as positive control.Panels B, D, and F: digital analysis of panels A, C, and E,respectively, demonstrating pixels above a fixed set threshold positivefor fluorescent intensity. Yellow arrows: immunoreactive junctionalplaques; red arrows: position of cell nuclei. Note the greater numberand intensity of junctional immunoreactive plaques in the culturestreated with the statistical mixture of stereoisomers of the disodiumsalt disuccinate astaxanthin derivative in comparison with solvent-onlytreated controls. The junctional plaques shown in Panels C and Drepresent infrequent plaques seen in controls; most cells in thesecultures were negative for Cx43 staining;

FIG. 17 depicts the 4 stereoisomers of the disodium disuccinate diesterof astaxanthin synthesized for the current studies (shown as the all-Egeometric isomers); the mixture of stereoisomers, or individualstereoisomers, were used in separate applications (see Figure legends);

FIG. 18 depicts the mean percent inhibition of superoxide anion signalas detected by DEPMPO spin trap by the disodium disuccinate derivativesof astaxanthin in pure aqueous formulation. Mixture=statistical mixtureof stereoisomers [3S,3′S, meso (3R,3′S and 3′R,3S), 3R,3′R in a 1:2:1ratio]Each derivative in aqueous formulation was standardized to controlEPR signal detected without addition of compound (set at 0% inhibitionby convention). Note the absence of superoxide inhibition by 3S,3′Sformulation in water. In each case, the aqueous formulation is lesspotent than the corresponding formulation in EtOH (FIG. 19);

FIG. 19 depicts the mean percent inhibition of superoxide anion signalas detected by DEPMPO spin trap by the disodium disuccinate derivativesof astaxanthin in ethanolic formulation. Mixture=statistical mixture ofstereoisomers [3S,3′S, meso (3R,3′S and 3′R,3S), 3R,3′R in a 1:2:1ratio]. The mixture, meso, and 3R, 3′R stock solutions were 1:2ethanol/water (33⅓% EtOH); the 3S,3′S stock solution was 1:1ethanol/water (50% EtOH). Final concentration of EtOH in the isolatedneutrophil test assay was 0.3% and 0.5%, respectively. Each derivativein ethanolic formulation was standardized to control EPR signal detectedwithout addition of compound (set at 0% inhibition by convention);

FIG. 20 depicts the mean percent inhibition of superoxide anion signalas detected by DEPMPO spin trap by the mixture of stereoisomers of thedisodium disuccinate derivative of astaxanthin (tested in 1:2 EtOH/waterformulation; final EtOH concentration in isolated neutrophil assay0.3%). As the concentration of the derivative increases, inhibitionincreases in a non-linear, dose-dependent manner. At 3 mM, near-completeinhibition of superoxide anion signal is seen (95.0% inhibition);

FIG. 21 depicts the mean percent inhibition of superoxide anion signalas detected by DEPMPO spin trap by the hydrochloride salt dilysineastaxanthin derivative. This derivative was highly water soluble (>50mg/mL), and did not require a co-solvent for excellent radical-quenchingability in this assay. Compare the superoxide anion inhibition of thisderivative with that depicted in FIG. 20, for a derivative that formssupramolecular assemblies in pure aqueous formulation;

FIG. 22 depicts a standard plot of concentration of non-esterified, freeastaxanthin versus time for plasma after single dose oral gavage inblack mice. Only non-esterified, free astaxanthin is detected in plasma,corroborating the complete de-esterification of the carotenoid analog inthe mammalian gut, as has been described previously;

FIG. 23 depicts a standard plot of concentration of non-esterified, freeastaxanthin verses time for liver after single dose oral gavage in blackmice. Only non-esterified, free astaxanthin is detected in liver, alsocorroborating (see FIG. 22 for plasma) the complete de-esterification ofthe carotenoid analog in the mammalian gut, as has been describedpreviously. At every time point, liver levels of non-esterified, freeastaxanthin are greater than that observed in plasma, a novel findingsuggesting vastly improved solid-organ delivery of free carotenoid inthe novel emulsion vehicle used in this study;

FIG. 24 depicts the effect of the disodium disuccinate astaxanthinderivative at 500 mg/kg by oral gavage on lipopolysaccharide(LPS)-induced liver injury in mice (as measured by elevation in serumalanine aminotransferase, or ALT). Three (3) animals were tested in eachgroup. Control animals received saline alone (sham-treated controls;left portion of figure) or emulsion without disodium disuccinateastaxanthin derivative (vehicle controls). Sham-treated animalsreceiving the novel derivative demonstrated no effect on backgroundlevels of ALT; mice receiving the oral emulsion with the novelderivative at 500 mg/kg showed reduced induced levels of ALT, indicatingprotection against hepatic necrosis after LPS insult;

FIG. 25 depicts a graphical representation of a relative reduction ofinfarct size in male Sprague-Dawley rats with pre-treatment using adisodium salt disuccinate astaxanthin derivative intravenous formulation(Cardax™). A linear relationship between dose and infarct size reductionwas seen. The levels of infarct size reduction approach that observedwith ischemic pre-conditioning;

FIG. 26 depicts a graphical representation of a relative reduction ofinfarct size in male Sprague-Dawley rats with pre-treatment using adisodium salt disuccinate astaxanthin derivative intravenous formulation(Cardax™);

FIG. 27 depicts transient absorption versus delay for the diaciddiscuccinate astaxanthin derivative (astaCOOH) using flash photolysis.The experiment was performed in acetonitrile (MeCN) using nitronaftalin(NN) as photosensitizer. The spectra obtained demonstrate that thediacid disuccinate astaxanthin derivative behaves identically tonon-esterified, free racemic astaxanthin as a radical quencher(formation of the carotenoid radical cation), identifying the derivativeas an active “soft-drug” which generates non-esterified, freeastaxanthin in vivo after both oral and intravenous delivery;

FIG. 28 depicts transient absorption versus delay for the referencecompound non-esterified, free racemic astaxanthin (asta)] using flashphotolysis. The experiment was performed in acetonitrile (MeCN) usingnitronaftalin (NN) as photosensitizer. The spectra obtained are nearlysuperimposable on those obtained for the diacid disuccinate astaxanthinderivative (astaCOOH), suggesting identical radical-cation formingproperties for both compounds;

FIG. 29 depicts a pictorial representation of a Western blot of apolyacrylamide gel with anti-connexin 43 antibody;

FIG. 30 depicts a pictorial representation of quantitative densitometricimages of Western blots with anti-connexin 43 antibodies followed by HRPchemiluminescence on a Biorad imager;

FIG. 31 depicts a graph of relative fold-induction of connexin 43expression by positive control (TTNPB, potent synthetic retinoid) andtest compounds (disodium salt disuccinate astaxanthin derivative in fourwater and/or ethanol (EtOH)/water formulations: H₂O-10-5, H₂O-10-6,H₂O-10-7, and EtOH/H₂O-10-5) versus sterile water control (H₂O) at 96hours post-dosing;

FIG. 32 depicts a graph of mean levels of non-esterified, freeastaxanthin in plasma and liver after eleven (11) days of oral gavage of500 mg/kg disodium disuccinate astaxanthin derivative (ADD) in emulsionvehicle to black mice. Both peak and trough levels in plasma and liverachieved were >200 nM, considered to be protective against oxidativestress and hepatic injury in vivo. The peak levels obtained in liver at6 hours post-11^(th) dose were nearly 9 times the protective levelsnecessary (1760 nM);

FIG. 33 depicts the mean percent inhibition of superoxide anion signalas detected by DEPMPO spin trap by the disodium salt disuccinatedi-vitamin C derivative [derivative (XXIII)]. As the concentration ofthe derivative increases, inhibition increases in a dose-dependentmanner. At 60 μM, nearly complete inhibition of superoxide anion signalis seen. This derivative was also highly water soluble, and wasintroduced into the test assay without a co-solvent (see FIG. 21). Thenovel derivative was comparable in radical-quenching efficacy to theformulation of the disodium salt disuccinate astaxanthin derivative in a1:2 formulation with vitamin C (see FIG. 3), suggesting active,“soft-drug” properties for this derivative. This co-antioxidantderivative strategy increased the relative radical scavenging potency(when compared with the disodium salt disuccinate astaxanthinderivative) by 50-fold;

FIG. 34 depicts effects of non-esterified, free astaxanthin (as theall-trans mixture of stereoisomers) on MCA-induced neoplastictransformation in mouse embryonic fibroblast (10T1/2) cells.Non-esterified, free astaxanthin is produced rapidly in vivo after oraland intravenous administration of novel carotenoid derivatives, and isdetected in high concentration in both plasma and solid organs (seeFIGS. 22 and 23). Non-esterified, free astaxanthin demonstrated levelsof reduction of neoplastic transformation (100%) above any othercarotenoid tested in this assay at similar concentrations, demonstratingthe increased utility of this compound for cancer chemopreventionapplications;

FIG. 35A-C depicts a comparison of an astaxanthin-treated dish tocontrol dishes (see description for FIG. 34);

FIG. 36 depicts a comparison of astaxanthin (as the mixture ofstereoisomers) to previously tested carotenoids in this laboratory usingthis assay (see description for FIG. 34);

FIG. 37 depicts a graphical representation of a relative reduction ofinfarct size in male New Zealand rabbits with pre-treatment using adisodium salt disuccinate astaxanthin derivative intravenous formulation(Cardax™). When compared with the infarct size reduction seen at thesame dose and identical pre-treatment schedule in rodents, a 38%increase in infarct size reduction was observed in the rabbit model; and

FIG. 38 depicts a graphical representation of a relative reduction ofcirculating levels of plasma C-reactive protein (CRP) in male NewZealand rabbits with pre-treatment using a disodium disuccinateastaxanthin derivative intravenous formulation (Cardax™). Controlrabbits (saline injection alone) stimulated for the acute-phase responsewith 1% croton oil by subcutaneous injection showed a mean increase of23.5% in circulating CRP levels from baseline (venous sample taken atthe time of reperfusion). In contrast, Cardax™—treated animals (50mg/kg) demonstrated a mean reduction in circulating CRP levels frombaseline (−15.7%), deomonstrating the potent anti-inflammatory effectsof Cardax™.

DETAILED DESCRIPTION

“Parent” carotenoids may generally refer to those natural compoundsutilized as starting scaffold for structural carotenoid analogsynthesis. Carotenoid derivatives may be derived from a naturallyoccurring carotenoid. Naturally occurring carotenoid may includelycopene, lycophyll, lycozanthin, astaxanthin, beta-carotene, lutein,zeaxanthin, and/or canthaxanthin to name a few.

Carotenoids are a group of natural pigments produced principally byplants, yeast, and microalgae. The family of related compounds nownumbers greater than 600 described members, exclusive of Z and Eisomers. Fifty (50) have been found in human sera or tissues. Humans andother animals cannot synthesize carotenoids de novo and must obtain themfrom their diet. All carotenoids share common chemical features, such asa polyisoprenoid structure, a long polyene chain forming thechromophore, and near symmetry around the central double bond.Tail-to-tail linkage of two C₂₀ geranylgeranyl diphosphate moleculesproduces the parent C₄₀ carbon skeleton. Carotenoids without oxygenatedfunctional groups are called “carotenes”, reflecting their hydrocarbonnature; oxygenated carotenes are known as “xanthophylls.” Cyclization atone or both ends of the molecule yields 7 identified end groups(representative structures shown in FIG. 1).

Documented carotenoid functions in nature include light-harvesting,photoprotection, and protective and sex-related coloration inmicroscopic organisms, mammals, and birds, respectively. A relativelyrecent observation has been the protective role of carotenoids againstage-related diseases in humans as part of a complex antioxidant networkwithin cells. This role is dictated by the close relationship betweenthe physicochemical properties of individual carotenoids and their invivo functions in organisms. The long system of alternating double andsingle bonds in the central part of the molecule (delocalizing theπ-orbital electrons over the entire length of the polyene chain) confersthe distinctive molecular shape, chemical reactivity, andlight-absorbing properties of carotenoids. Additionally, isomerismaround C═C double bonds yields distinctly different molecular structuresthat may be isolated as separate compounds (known as Z (“cis”) and E(“trans”), or geometric, isomers). Of the more than 600 describedcarotenoids, an even greater number of the theoretically possible mono-Zand poly-Z isomers are sometimes encountered in nature. The presence ofa Z double bond creates greater steric hindrance between nearby hydrogenatoms and/or methyl groups, so that Z isomers are generally less stablethermodynamically, and more chemically reactive, than the correspondingall-E form. The all-E configuration is an extended, linear, and rigidmolecule. Z-isomers are, by contrast, not simple, linear molecules (theso-called “bent-chain” isomers). The presence of any Z in the polyenechain creates a bent-chain molecule. The tendency of Z-isomers tocrystallize or aggregate is much less than all-E, and Z isomers are morereadily solubilized, absorbed, and transported in vivo than their all-Ecounterparts. This has important implications for enteral (e.g., oral)and parenteral (e.g., intravenous, intra-arterial, intramuscular, andsubcutaneous) dosing in mammals.

Carotenoids with chiral centers may exist either as the R (rectus) or S(sinister) configurations. As an example, astaxanthin (with 2 chiralcenters at the 3 and 3′ carbons) may exist as 4 possible stereoisomers:3S, 3′S; 3R, 3′S and 3S, 3′R (meso forms); or 3R, 3′R. The relativeproportions of each of the stereoisomer may vary by natural source. Forexample, Haematococcus pluvialis microalgal meal is 99% 3S, 3′Sastaxanthin, and is likely the predominant human evolutionary source ofastaxanthin. Krill (3R,3′R) and yeast sources yield differentstereoisomer compositions than the microalgal source. Syntheticastaxanthin, produced by large manufacturers such as Hoffmann-LaRocheAG, Buckton Scott (USA), or BASF AG, are provided as defined geometricisomer mixtures of a 1:2:1 stereoisomer mixture [3S, 3′S; 3R, 3′S,3′R,3S (meso); 3R, 3′R] of non-esterified, free astaxanthin. Naturalsource astaxanthin from salmonid fish is predominantly a singlestereoisomer (3S,3′S), but does contain a mixture of geometric isomers.Astaxanthin from the natural source Haematococcus pluvialis may containnearly 50% Z isomers. As stated above, the Z conformational change maylead to a higher steric interference between the two parts of thecarotenoid molecule, rendering it less stable, more reactive, and moresusceptible to reactivity at low oxygen tensions. In such a situation,in relation to the all-E form, the Z forms: (1) may be degraded first;(2) may better suppress the attack of cells by reactive oxygen speciessuch as superoxide anion; and (3) may preferentially slow the formationof radicals. Overall, the Z forms may initially be thermodynamicallyfavored to protect the lipophilic portions of the cell and the cellmembrane from destruction. It is important to note, however, that theall-E form of astaxanthin, unlike β-carotene, retains significant oralbioavailability as well as antioxidant capacity in the form of itsdihydroxy- and diketo-substitutions on the β-ionone rings, and has beendemonstrated to have increased efficacy over β-carotene in most studies.The all-E form of astaxanthin has also been postulated to have the mostmembrane-stabilizing effect on cells in vivo. Therefore, it is likelythat the all-E form of astaxanthin in natural and synthetic mixtures ofstereoisomers is also extremely important in antioxidant mechanisms, andmay be the form most suitable for particular pharmaceuticalpreparations.

The antioxidant mechanism(s) of carotenoids, and in particularastaxanthin, includes singlet oxygen quenching, direct radicalscavenging, and lipid peroxidation chain-breaking. The polyene chain ofthe carotenoid absorbs the excited energy of singlet oxygen, effectivelystabilizing the energy transfer by delocalization along the chain, anddissipates the energy to the local environment as heat. Transfer ofenergy from triplet-state chlorophyll (in plants) or other porphyrinsand proto-porphyrins (in mammals) to carotenoids occurs much morereadily than the alternative energy transfer to oxygen to form thehighly reactive and destructive singlet oxygen (¹O₂). Carotenoids mayalso accept the excitation energy from singlet oxygen if any should beformed in situ, and again dissipate the energy as heat to the localenvironment. This singlet oxygen quenching ability has significantimplications in cardiac ischemia, macular degeneration, porphyria, andother disease states in which production of singlet oxygen has damagingeffects. In the physical quenching mechanism, the carotenoid moleculemay be regenerated (most frequently), or be lost. Carotenoids are alsoexcellent chain-breaking antioxidants, a mechanism important ininhibiting the peroxidation of lipids. Astaxanthin can donate a hydrogen(H) to the unstable polyunsaturated fatty acid (PUFA) radical, stoppingthe chain reaction. Peroxyl radicals may also, by addition to thepolyene chain of carotenoids, be the proximate cause for lipid peroxidechain termination. The appropriate dose of astaxanthin has been shown tocompletely suppress the peroxyl radical chain reaction in liposomesystems. Astaxanthin shares with vitamin E this dual antioxidant defensesystem of singlet oxygen quenching and direct radical scavenging, and inmost instances (and particularly at low oxygen tension in vivo) issuperior to vitamin E as a radical scavenger and physical quencher ofsinglet oxygen.

Carotenoids, and in particular astaxanthin, are potent direct radicalscavengers and singlet oxygen quenchers and possess all the desirablequalities of such therapeutic agents for inhibition or amelioration ofreperfusion injury. Synthesis of novel carotenoid derivatives with“soft-drug” properties (i.e. activity in the derivatized form), withphysiologically relevant, cleavable linkages to pro-moieties, cangenerate significant levels of free carotenoids in both plasma and solidorgans. In the case of non-esterified, free astaxanthin, this is aparticularly useful embodiment (characteristics specific tonon-esterified, free astaxanthin below):

-   -   Lipid soluble in natural form; may be modified to become more        water soluble    -   Molecular weight of 597 Daltons [size <600 daltons (Da) readily        crosses the blood brain barrier, or BBB]    -   Long polyene chain characteristic of carotenoids effective in        singlet oxygen quenching and lipid peroxidation chain breaking    -   No pro-vitamin A activity in mammals (eliminating concerns of        hypervitaminosis A and retinoid toxicity in humans).

The administration of antioxidants which are potent singlet oxygenquenchers and direct radical scavengers, particularly of superoxideanion, should limit hepatic fibrosis and the progression to cirrhosis byaffecting the activation of hepatic stellate cells early in thefibrogenetic pathway. Reduction in the level of ROS by theadministration of a potent antioxidant can therefore be crucial in theprevention of the activation of both HSC and Kupffer cells. Thisprotective antioxidant effect appears to be spread across the range ofpotential therapeutic antioxidants, including water-soluble (e.g.,vitamin C, glutathione, resveratrol) and lipophilic (e.g., vitamin E,β-carotene, astaxanthin) agents. Therefore, a co-antioxidant derivativestrategy in which water-soluble and lipophilic agents are combinedsynthetically is a particularly useful embodiment.

Vitamin E is generally considered the reference antioxidant. Whencompared with vitamin E, carotenoids are more efficient in quenchingsinglet oxygen in homogenenous organic solvents and in liposome systems.They are better chain-breaking antioxidants as well in liposomalsystems. They have demonstrated increased efficacy and potency in vivo.They are particularly effective at low oxygen tension, and in lowconcentration, making them extremely effective agents in diseaseconditions in which ischemia is an important part of the tissue injuryand pathology. These carotenoids also have a natural tropism for theliver after oral administration. Therefore, therapeutic administrationof carotenoids should provide a greater benefit in limiting fibrosisthan vitamin E.

Problems related to the use of some carotenoids and structuralcarotenoid analogs include: (1) the complex isomeric mixtures, includingnon-carotenoid contaminants, provided in natural and synthetic sourcesleading to costly increases in safety and efficacy tests required bysuch agencies as the FDA; (2) limited bioavailability uponadministration to a subject; and (3) the differential induction ofcytochrome P450 enzymes (this family of enzymes exhibitsspecies-specific differences which must be taken into account whenextrapolating animal work to human studies).

In an embodiment, the parent carotenoid may have a structure of anynaturally occurring carotenoid. Some examples of naturally occurringcarotenoids that may be used as parent compounds are shown in FIG. 1.

In some embodiments, the carotenoid derivatives may include compoundshaving the structure (I):

Each R³ may be independently hydrogen, methyl, alkyl, alkenyl, oraromatic substituents. R¹ and R² may be independently H, an acyclicalkene with at least one substituent, or a cyclic ring with at least onesubstituent having general structure (II):

where n may be between 4 to 10 carbon atoms. W is the substituent. Thesubstituent may be at least partially hydrophilic. A hydrophilicsubstituent may assist in increasing the water solubility of acarotenoid derivative. In some embodiments, a carotenoid derivative maybe at least partially water soluble. The cyclic ring may include atleast one chiral center. The acyclic alkene may include at least onechiral center. The cyclic ring may include at least one degree ofunsaturation. In some cyclic ring embodiments, the cyclic ring may bearomatic. The cyclic ring may include a substituent. The substituent maybe hydrophilic. In some embodiments, the cyclic ring may include, forexample (a), (b), or (c):

In some embodiments, the substituent may include, for example, acarboxylic acid, an amino acid, an ester, an alkanol, an amine, aphosphate, a succinate, a glycinate, an ether, a glucoside, a sugar, ora carboxylate salt.

In some embodiments, each substituent—W may independently include—XR.Each X may independently include O, N, or S. In some embodiments, eachsubstituent—W may independently comprises amino acids, esters,carbamates, amides, carbonates, alcohol, phosphates, or sulfonates. Insome substituent embodiments, the substituent may include, for example(d) through (pp):

where each R is, for example, independently -alkyl-NR¹ ₃ ⁺,-aromatic-NR¹ ₃ ⁺, -alkyl-CO₂ ⁻, -aromatic-CO₂ ⁻, -amino acid-NH₃ ⁺,-phosphorylated amino acid-NH₃ ⁺, polyethylene glycol, dextran, H,alkyl, or aryl. In some embodiments, substituents may include anycombination of (d) through (pp). In some embodiments, negatively chargedsubstituents may include alkali metals, one metal or a combination ofdifferent alkali metals in an embodiment with more than one negativelycharged substituent, as counter ions. Alkali metals may include, but arenot limited to, sodium, potassium, and/or lithium.

Although the above structure, and subsequent structures, depict alkenesin the E configuration this should not be seen as limiting. Compoundsdiscussed herein may include embodiments where alkenes are in the Zconfiguration or include alkenes in a combination of Z and Econfigurations within the same molecule. The compounds depicted hereinmay naturally convert between the Z and E configuration and/or exist inequilibrium between the two configurations.

In an embodiment, a chemical compound may include a carotenoidderivative having the structure (III)

Each Y may be independently O or H₂. Each R may be independently OR¹ orR¹.Each R¹ may be independently -alkyl-NR² ₃ ⁺, -aromatic-NR² ₃ ⁺,-alkyl-CO₂ ⁻, -aromatic-CO₂ ⁻, -amino acid-NH₃ ⁺, -phosphorylated aminoacid-NH₃ ⁺, polyethylene glycol, dextran, H, alkyl, peptides,poly-lysine or aryl. In addition, each R² may be independently H, alkyl,or aryl. The carotenoid derivative may include at least one chiralcenter.

In a specific embodiment where Y is H₂, the carotenoid derivative hasthe structure (IV)

In a specific embodiment where Y is O, the carotenoid derivative has thestructure (V)

In an embodiment, a chemical compound may include a carotenoidderivative having the structure (VI)

Each Y may be independently O or H₂. Each R may be independently H,alkyl, or aryl. The carotenoid derivative may include at least onechiral center. In a specific embodiment Y may be H₂, the carotenoidderivative having the structure (VII)

In a specific embodiment where Y is O, the carotenoid derivative has thestructure (VIII)

In an embodiment, a chemical compound may include a carotenoidderivative having the structure (IX)

Each Y may be independently O or H₂. Each R′ may be CH₂. n may be 1 to9. Each X may be independently

Each R may be independently -alkyl-NR¹ ₃ ⁺, -aromatic-NR¹ ₃ ⁺,-alkyl-CO₂ ⁻, -aromatic-CO₂ ⁻, -amino acid-NH₃ ⁺, -phosphorylated aminoacid-NH₃ ⁺, polyethylene glycol, dextran, H, alkyl, or aryl. Each R¹ maybe independently H, alkyl, or aryl. The carotenoid derivative mayinclude at least one chiral center.

In a specific embodiment where Y is H₂, the carotenoid derivative hasthe structure (X)

In a specific embodiment where Y is O, the carotenoid derivative has thestructure (XI)

In an embodiment, a chemical compound may include a carotenoidderivative having the structure (XII)

Each Y may be independently O or H₂. The carotenoid derivative mayinclude at least one chiral center. In a specific embodiment Y may beH₂, the carotenoid derivative having the structure (XIII)

In a specific embodiment where Y is O, the carotenoid derivative has thestructure (XIV)

In some embodiments, a chemical compound may include a disuccinic acidester carotenoid derivative having the structure (XV)

In some embodiments, a chemical compound may include a disodium saltdisuccinic acid ester carotenoid derivative having the structure (XVI)

In some embodiments, a chemical compound may include a carotenoidderivative with a co-antioxidant, in particular one or more analogs ofvitamin C (i.e., L ascorbic acid) coupled to a carotenoid. Someembodiments may include carboxylic acid and/or carboxylate derivativesof vitamin C coupled to a carotenoid (e.g., structure (XVII))

Carbohydr. Res. 1978, 60, 251-258, herein incorporated by reference,discloses oxidation at C-6 of ascorbic acid as depicted in EQN. 5.

Some embodiments may include vitamin C and/or vitamin C analogs coupledto a carotenoid. Vitamin C may be coupled to the carotenoid via an etherlinkage (e.g., structure (XVIII))

Some embodiments may include vitamin C disuccinate analogs coupled to acarotenoid (e.g., structure (XIX)

Some embodiments may include solutions or pharmaceutical preparations ofcarotenoids and/or carotenoid derivatives combined with co-antioxidants,in particular vitamin C and/or vitamin C analogs. Pharmaceuticalpreparations may include about a 2:1 ratio of vitamin C to carotenoidrespectively.

In some embodiments, a carotenoid (e.g., astaxanthin) may be coupled tovitamin C forming an ether linkage. The ether linkage may be formedusing the Mitsunobu reaction as in EQN. 1.

In some embodiments, vitamin C may be selectively esterified. Vitamin Cmay be selectively esterified at the C-3 position (e.g., EQN. 2). J.Org. Chem. 2000, 65, 911-913, herein incorporated by reference,discloses selective esterification at C-3 of unprotected ascorbic acidwith primary alcohols.

In some embodiments, a carotenoid may be coupled to vitamin C. Vitamin Cmay be coupled to the carotenoid at the C-6, C-5 diol position asdepicted in EQNS. 3 and 4 forming an acetal.

In some embodiments, a carotenoid may be coupled to a water solublemoiety (e.g., vitamin C) with a glyoxylate linker as depicted in EQN. 6.Tetrahedron 1989, 22, 6987-6998, herein incorporated by reference,discloses similar acetal formations.

In some embodiments, a carotenoid may be coupled to a water solublemoiety (e.g., vitamin C) with a glyoxylate linker as depicted in EQN. 7.J. Med. Chem. 1988, 31, 1363-1368, herein incorporated by reference,discloses the glyoxylic acid chloride.

In some embodiments, a carotenoid may be coupled to a water solublemoiety (e.g., vitamin C) with a phosphate linker as depicted in EQN. 8.Carbohydr. Res. 1988, 176, 73-78, herein incorporated by reference,discloses the L-ascorbate 6-phosphate.

In some embodiments, a carotenoid may be coupled to a water solublemoiety (e.g., vitamin C) with a phosphate linker as depicted in EQN. 9.Carbohydr. Res. 1979, 68, 313-319, herein incorporated by reference,discloses the 6-bromo derivative of vitamin C. Carbohydr. Res. 1988,176, 73-78, herein incorporated by reference, discloses the 6-bromoderivative of vitamin C's reaction with phosphates.

In some embodiments, a carotenoid may be coupled to a water solublemoiety (e.g., vitamin C) with a phosphate linker as depicted in EQN. 10.J. Med Chem. 2001, 44, 1749-1757 and J. Med Chem. 2001, 44, 3710-3720,herein incorporated by reference, disclose the allyl chloride derivativeand its reaction with nucleophiles, including phosphates, under mildbasic conditions.

In some embodiments, a carotenoid may be coupled to a water solublemoiety (e.g., vitamin C) with a phosphate linker as depicted in EQN. 11.Vitamin C may be coupled to the carotenoid using selectiveesterification at C-3 of unprotected ascorbic acid with primaryalcohols.

In some embodiments, a chemical compound may include a carotenoidderivative including one or more amino acids (e.g., lysine) and/or aminoacid analogs (e.g., lysine hydrochloric acid salt) coupled to acarotenoid [e.g., structure (XX)].

In some embodiments, a carotenoid derivative may include:

In an embodiment, the carotenoid derivatives may be synthesized fromnaturally occurring carotenoids. The carotenoids may include structures2A-2E depicted in FIG. 1. In some embodiments, the carotenoidderivatives may be synthesized from a naturally occurring carotenoidincluding one or more alcohol substituents. In other embodiments, thecarotenoid derivatives may be synthesized from a derivative of anaturally occurring carotenoid including one or more alcoholsubstituents. The synthesis may result in a single stereoisomer. Thesynthesis may result in a single geometric isomer of the carotenoidderivative. The synthesis/synthetic sequence may include any priorpurification or isolation steps carried out on the parent carotenoid. Anexample may include, but is not limited to, a 3S,3′S all-E carotenoidderivative, where the parent carotenoid is astaxanthin. The syntheticsequence may include protecting and subsequently deprotecting variousfunctionalities of the carotenoid and/or substituent precursor. Thealcohols may be deprotonated with a base. The deprotonated alcohol maybe reacted with a substituent precursor with a good leaving group. Thebase may include any non-nucleophilic base known to one skilled in theart such as, for example, dimethylaminopyridine. The deprotonatedalcohol may act as a nucleophile reacting with the substituentprecursor, displacing the leaving group. Leaving goups may include, butare not limited to, Cl, Br, tosyl, brosyl, mesyl, or trifyl. These areonly a few examples of leaving groups that may be used, many more areknown and would be apparent to one skilled in the art. In someembodiments, it may not even be necessary to deprotonate the alcohol,depending on the leaving group employed. In other examples the leavinggroup may be internal and may subsequently be included in the finalstructure of the carotenoid derivative, a non-limiting example mayinclude anhydrides or strained cyclic ethers. For example, thedeprotonated alcohol may be reacted with succinic anhydride. In anembodiment, the disuccinic acid ester of astaxanthin may be furtherconverted to the disodium salt. Examples of synthetic sequences for thepreparation of some of the specific embodiments depicted are describedin the Examples section. The example depicted below is a genericnon-limiting example of a synthetic sequence for the preparation ofcarotenoid derivatives.

Ischemia-Reperfusion (L/R) Injury: Pathophysiologic Features

Reperfusion of ischemic myocardium results in significant cellular andlocal alterations in at-risk tissue which exacerbate damage created bythe ischemic insult. Specifically, vascular and microvascular injury,endothelial dysfunction, accelerated cellular necrosis, and granulocyteactivation occur subsequent to reperfusion. Vascular and microvascularinjury results from complement activation, the interaction ofcirculating and localized C-reactive protein with Clq and phosphocholineon exposed cells forming the membrane attack complex (MAC) with ensuingcell death and increased endothelial permeability, superoxide anion (O₂⁻ ) generation by affected endothelium and activated leukocytes,microemboli, cytokine release (in particular IL-6), and activation ofplatelets with IIbIIIa receptor activation, and subsequent release ofADP and serotonin. Endothelial dysfunction follows, with subsequentgeneration of superoxide anion by the dysfunctional endothelium, furtherdamaging the affected endothelium in a positive feedback cycle. It hasbeen shown that ischemia-reperfusion results in early and severe injuryto the vasculature, which further compromises myocyte survival.Granulocyte activation also occurs during reperfusion. The activationand degranulation of this cell lineage results in the release ofmyeloperoxidase (MPO), elastases, proteases, and oxygen-derived radicaland non-radical species (most importantly superoxide anion,hypochlorite, singlet oxygen, and hydrogen peroxide after the“respiratory burst”). Oxygen-derived radical and non-radical (e.g.singlet oxygen) species are implicated in much of the damage associatedwith ischemia and reperfusion, and lipid peroxidation has clearly beenshown to be a sequel of reperfusion as measured by thiobarbituric acidreactive substances (TBARS), malondialdehyde (MDA), or conjugated dieneformaton.

The ischemic insult to both the endothelium of coronary vessels and themyocardium itself creates conditions favoring the production of radicalsand other non-radical oxygen-derived species capable of damaging tissueherein collectively referred to as reactive oxygen species (“ROS”). Theendothelium-based xanthine dehydrogenase—xanthine oxidase system inhumans is a source of the superoxide anion (O₂ ⁻ ). The human myocardiumlacks this enzyme system. In healthy tissue, 90% of the enzyme exists asthe dehydrogenase (D) form; it is converted to the oxidase (O) form inischemic tissue. The (O)-form, using molecular oxygen as the electronacceptor, produces the superoxide anion O₂ ⁻ in the coronaryendothelium. Superoxide anion is then available to create additionaltissue damage in the local environment. The superoxide anion is not themost reactive or destructive radical species in biological systems onits own. However, it is the source of some shorter- and longer-lived,more damaging radicals and/or ROS such as the hydroxyl radical, hydrogenperoxide, singlet oxygen, and peroxyl radicals. As such, it can beconsidered the “lynchpin” radical in I/R injury. The biologicalreactions of the superoxide radical to form these important oxidants areshown below:

-   (1) superoxide anion may accept a single electron (“monovalent    reduction”), producing peroxide (O₂ ⁻²). Coupled with 2 protons,    peroxide then forms hydrogen peroxide (H₂O₂). H₂O₂ diffuses easily    through cell membranes and cannot readily be excluded from the    cytoplasm, where it may react with cellular components or activate    central inflammatory cascades such as nuclear factor kappa-B    (NF-kappa-B), which are also implicated in the additional    inflammatory damage in I/R injury.-   (2) superoxide anion typically reacts with itself to produce    hydrogen peroxide and oxygen (“dismutation”). Superoxide dismutation    may be spontaneous, or catalyzed by the enzyme superoxide dismutase    (SOD), a reaction which results in the formation of oxidized SOD:    2O₂ ⁻+2H⁺→H₂O₂+³O₂-   (3) superoxide anion may serve as a reducing agent and donate a    single electron (“monovalent reduction”) to a metal cation. For    example, in the two step process below, ferric iron (Fe³⁺) is    reduced and subsequently acts as a catalyst to convert hydrogen    peroxide (H₂O₂) into the hydroxyl radical (HO.).    O₂ ⁻+Fe³⁺→³O₂+Fe²⁺  (step 1)-   Ferrous iron (Fe²⁺), the reduced metal cation, subsequently    catalyzes the breaking of the oxygen-oxygen bond of hydrogen    peroxide. This produces one hydroxyl radical (HO.) and one hydroxide    ion (HO⁻). The reaction is known as the Fenton reaction,    particularly important in reperfusion injury where iron and/or    copper compartmentalization has been lost (typically through    hemolysis of red blood cells, RBCs):    Fe²⁺+H₂O₂→Fe³⁺+HO.+HO⁻  (step 2)    Hydroxyl radicals readily cross cellular membranes. Hydroxyl radical    damage is “diffusion rate-limited”, that is, the 3-dimensional    distance in which damage may be inflicted is related to the    radical's rate of diffusion. The hydroxyl radical is a particularly    toxic ROS. Hydroxyl radicals may add to organic substrates    (represented by R in the reaction below) and form a hydroxylated    adduct which is itself a radical. In the case of    ischemia-reperfusion injury, polyunsaturated fatty acids (PUFAs) in    endothelial and myocyte membranes are particularly susceptible to    hydroxyl radical damage:    HO.+R→HOR. (hydroxylated adduct)    The adduct formed above may further oxidize in the presence of metal    cations or molecular oxygen. This results in oxidized, stable    product(s). In the first case, the extra electron is transferred to    the metal ion, and in the second case, to oxygen (forming    superoxide). Two adduct radicals may also react with each other    forming oxidized, stable, and crosslinked products plus water. This    is an important process in the oxidation of membrane proteins:    HOR.+HOR.→R—R.+2H₂O    In addition, hydroxyl radicals may oxidize organic substrates by    abstracting electrons from such molecules:    HO.+R→R.+OH⁻    The oxidized substrate (R.) is a radical. Such radicals may react    with other molecules in a chain reaction. Carotenoids are    particularly efficient lipid-peroxidation chain breakers. In one    instance, the reaction with ground-state oxygen produces peroxyl    radicals (ROO.):    R.+³O₂→ROO.    Peroxyl radicals are very reactive. They may react with other    organic substrates in a chain reaction:    ROO.+RH→ROOH+R.    Chain reactions are common in the oxidative damage of PUFAs and    other susceptible membrane lipids. Measurement of the rate of oxygen    consumption is one indication of the initiation and progress of the    chain reaction. It is important to note that, in liposomal model    systems, non-esterified, free astaxanthin at the appropriate dose is    capable of complete suppression of the chain reaction and    accompanying oxygen consumption.-   (4) superoxide anion may react with the hydroxyl radical (HO.) to    form singlet oxygen (¹O₂*). Singlet oxygen is not a radical, but is    highly reactive and damaging in cardiac biological systems. Singlet    oxygen has been implicated in the destruction of membrane-bound    proteins such as 5′-nucleotidase, important in the maintenance or    restoration of local concentrations of vasodilatory compounds such    as adenosine (shown to be effective in humans for reduction of    infarct size):    O₂ ⁻+HO.→¹O₂* +HO⁻-   (5) superoxide anion may also react with the radical nitric oxide    (NO.), producing peroxynitrite (OONO⁻). Peroxynitrite is a highly    reactive and damaging molecule in biological systems.    O₂ ⁻+NO.→OONO⁻

Polymorphonuclear leukocytes (PMNs), in particular neutrophils, andactivated macrophages are a rich source of oxygen-derived radical andnon-radical species. The NADPH-oxidase system located in phagocyte cellmembranes is an important source of radicals following stimulation. ThePMNs and activated macrophages rapidly consume oxygen in the“respiratory burst” and convert it to superoxide anion and subsequentlyhydrogen peroxide (H₂O₂), as well as significant amounts of singletoxygen. PMNs are additionally a source of hypochlorite, another damagingreactive oxygen species. While important in phagocytic cell activity ininfection, in the local environment during ischemia and reperfusion,further cellular injury occurs as these ROS attack normal and damagedhost cells in the local area.

Neutrophils are a primary source of oxygen radicals during reperfusionafter prolonged myocardial ischemia, particularly in animal models ofexperimental infarction. Many prior studies have documented oxygenradical formation during ischemia-reperfusion, but few addressed thesource(s) of such radicals in vivo, or had examined radical generationin the context of prolonged myocardial ischemia. Neutrophils arerecruited in large amounts within the previously ischemic tissue and arethought to induce injury by local release of various mediators, chieflyoxygen radicals. Previously, the contribution of activated neutrophilsto reperfusion injury and potential myocardial salvage remained unclear.A methodology was developed to detect radicals, in particular superoxideanion, without interfering with the blood-borne mechanisms of radicalgeneration.

Open- and closed-chest dogs underwent aorta and coronary sinuscatheterization (Duilio et al. 2001). No chemicals were infused.Instead, blood was drawn into syringes pre-filled with a spin trap andanalyzed by electron paramagnetic resonance (EPR) spectroscopy. After 90minutes of coronary artery occlusion, the transcardiac concentration ofoxygen radicals rose several-fold 10 minutes after reflow, and remainedsignificantly elevated for at least 1 hour. Radicals were mostly derivedfrom neutrophils, in particular superoxide anion. These radicalsexhibited marked reduction after the administration of (1) neutrophilNADPH-oxidase inhibitors and (2) a monoclonal antibody (R15.7) againstneutrophil CD18-adhesion molecule. The first intervention was designedto reduce the neutrophil respiratory burst, and the second to reducerecruitment of neutrophils to the site(s) of reperfusion injury. Thereduction of radical generation by the monoclonal antibody R15.7 wasalso associated with a significantly smaller infarct size and with aconcomitant decrease in no-reflow areas. It was demonstrated for thefirst time that activated neutrophils were a major source of oxidants inhearts reperfused in vivo after prolonged ischemia, that this phenomenonwas long-lived, and that anti-neutrophil interventions could effectivelyprevent the increase in transcardiac concentration of oxygen radicalsduring reperfusion. In these animal models of experimental infarction,the lack of pre-existing pathology prior to coronary artery occlusionmay over-emphasize the contribution of neutrophilic recruitment andactivation to I/R injury; indeed, in the human atheroscleroticsituation, activated macrophages and activated T-lymphocytes alreadyresiding in the “area-at-risk” may also contribute substantially to I/Rinjury.

Ischemia causes depletion of ATP in cells in the affected area. At thelevel of the mitochondrial electron transport chain, which normally“leaks” approximately 5% of the processed electrons in healthy tissue,further leakage of partially-reduced oxygen species (in particular O₂ ⁻) is favored when the respiratory chain becomes largely reduced. Thishappens primarily during ischemia. The net effect in the local cellularenvironment is a tip in the balance of the redox status fromanti-oxidant to pro-oxidant, which is at the same time less capable ofabsorbing additional radical insult(s) without further cellular damage.

Prevention of Ischemia-Reperfusion Injury: Pharmacologic Agents Used inPrevious Animal and/or Human Trials

The following compounds have been evaluated, either in animal models orin limited human trials, as therapeutic agents for the reduction ofischemia-reperfusion injury and/or myocardial salvage during acutemyocardial infarction (AMI). Most are biological antioxidants.

-   -   Superoxide dismutase (and derivatives or mimetics)    -   Catalase    -   Glutathione and glutathione peroxidase    -   Xanthine oxidase inhibitors    -   Vitamins B, C, E (and derivatives)    -   Calcium antagonists    -   ACE inhibitors    -   Sulphydryl thiol compounds (in particular N-acetylcysteine)    -   Iron chelators (desferioxamine)    -   Anti-inflammatories (e.g., ibuprofen)    -   Phosphocreatine    -   N-2-mercaptopropionyl glycine (MPG)    -   Probucol (and derivatives)    -   Melatonin    -   Coenzyme Q-10

Seminal work by Singh and co-workers in India previously demonstratedthat human patients presenting with acute myocardial infarction aredepleted in endogenous antioxidants, and that supplementation withantioxidant cocktails and/or monotherapy with coenzyme Q10 (a potentlipophilic antioxidant) were useful to achieve both myocardial salvageand improvement in traditional hard clinical endpoints (such as totalcardiac deaths and nonfatal reinfarction) at 30 days post-AMI. TheAMISTAD trial demonstrated the usefulness of adenosine as a myocardialsalvage agent in 3 separate groups of patients. RheothRx™ (a rheologicalagent) was also efficacious as a salvage agent in human trials, but wasabandoned secondary to renal toxicity. Most recently, Medicure, Inc.demonstrated the utility of a vitamin B derivative for myocardialsalvage in a small Phase II pilot study in collaboration with the DukeClinical Research Institute. Hence, the “translational” problem (fromefficacy in animal models of experimental infarction to human clinicalefficacy) identified in previous reviews of I/R injury is now betterunderstood. However, the commercial window-of-opportunity still exists,as no agent has been specifically approved for human use as a salvageagent.

Timing of Treatment For Myocardial Ischemia-Reperfusion Injury

As discussed above, early reperfusion of acute myocardial infarctions(primarily with pharmacological or surgical reperfusion) halts celldeath due to ischemia, but paradoxically causes further injury—mostlikely by oxidant mechanisms. Horwitz et al. (1999) identified thewindow of opportunity during which antioxidants must be present intherapeutic concentrations to prevent reperfusion injury during 90minutes of ischemia, and 48 hours of subsequent reperfusion, in 57 dogs.Statistical analyses in the trial focused on identifying components ofgroup membership responsible for differences in infarct size, andrevealed that duration of treatment was a major determinant. If begun atany time within the first hour of reperfusion, infusions of greater thanor equal to 3 hours markedly diminished infarct size. Duilio et al.(2001) further clarified this issue by demonstrating that oxygenconsumption reflective of the peroxyl radical chain reaction begins 10minutes after reperfusion, and that radical activity remains elevatedfor at least the first hour of reperfusion in a canine model. Singh etal. (1996) previously demonstrated in human patients that myocardialsalvage, and improvement of hard clinical endpoints (nonfatalreinfarction, death) was possible starting antioxidant therapy onaverage 13 hours post-MI, and continuing for 28 days. Therefore, plasmaantioxidants with long half-lives may be particularly appropriate forthis setting, as they may be administered as a loading dose and allowedto decay in the plasma throughout the critical early post-AMI period (0to 24 hours). The plasma half-lives of carotenoids administered orallyrange from approximately 21 hours for the xanthophylls (“oxygenated”carotenoids including astaxanthin, capsanthin, lutein, and zeaxanthin)to 222 hours for carotenes (“hydrocarbon” carotenoids such as lycopene).The significant difference in plasma antioxidant half-life (7 minutes)in the trial by Horwitz et al. (1999), for superoxide dismutase and itsmimetics in human studies, versus a nearly 21 hour half-life forxanthophylls and nearly 9 days for carotenes, highlights thepharmacokinetic advantages and potential cardioprotection against I/Rinjury by carotenoids in AMI in humans.

Critical Appraisal of Antioxidants in Reperfusion Injury: Human Studies

Mean levels of vitamins A, C, E, and β-carotene were significantlyreduced in patients presenting with AMI, compared with control patientsin a study conducted by Singh et al. (1994). Lipid peroxides weresignificantly elevated in the AMI patients. The inverse relationshipbetween AMI and low plasma levels of vitamins remained significant afteradjustment for smoking and diabetes in these patients. Similarly, 38patients with AMI were studied by Levy et al. (1998), and exhibitedsignificantly decreased levels of vitamins A, E, and β-carotene comparedwith age-matched, healthy control subjects. After thrombolysis, lipidperoxidation products increased significantly in the serum of treatedpatients. Thrombolytic therapy also caused a significant decrease inplasma vitamin E levels. These descriptive studies indicate that uponpresentation with AMI, it is likely that serum levels of antioxidantvitamins will be decreased in patients undergoing an acute coronaryevent. Pharmacologic intervention with antioxidant compounds in theacute setting would likely remedy deficiencies in antioxidant vitaminsand total body antioxidant status.

Prospective human intervention trials with antioxidants in the settingof primary and/or secondary prevention of CVD are similarly limited, buthave been largely successful. Four out of five recent human studiesstrongly support the effectiveness of vitamin E in reducing heartdisease risk and complication rates. The Secondary Prevention withAntioxidants of Cardiovascular Disease in End-Stage Renal Disease study,in patients with significant kidney disease, revealed a 70% reduction innonfatal MI in patients given 800 IU per day of natural source vitaminE. Similarly, as mentioned herein, a number of agents have now beensuccessfully applied to myocardial salvage applications in humans.

Delivery of a low molecular weight compound intravenously in the acutesetting to inhibit or ameliorate I/R injury will require an evaluationof its immunogenicity. The incidence of transfusion-type and otheradverse reactions to the rapid infusion of the compound must beminimized. Compounds with a molecular weight <1000 Da, e.g. aspirin,progesterone, and astaxanthin, are likely not immunogenic unlesscomplexed with a carrier. As molecular weight increases to between 1000and 6000 Da, e.g. insulin and ACTH, the compound may or may not beimmunogenic. As molecular weight increases to >6000 Da, the compound islikely to be immunogenic. In addition, lipids are rarely immunogenic,again unless complexed to a carrier. Astaxanthin, as a xanthophyllcarotenoid, is highly lipid soluble in natural form. It is also small insize (597 Da). Therefore, an injectable astaxanthin structural analoghas a low likelihood of immunogenicity in the right formulation, and isa particularly desirable compound for the current therapeuticindication.

Prevention of Arrhythmia: Pharmacologic Agents Used in Previous AnimalTrials

Studies conducted by Gutstein et al. (2001) evaluated geneticallymodified mice incapable of expressing connexin 43 in the myocardium[Cx43 conditional knockout (CKO) mice]. Gutstein et al. discovered thatdespite normal heart structure and contractile performance, Cx43 CKOmice uniformly developed sudden cardiac death, apparently fromspontaneous ventricular lethal tachycardia(s). This data supports thecritical role of the gap junction channel, and connexin 43 inparticular, in maintaining cardiac electrical stability. Connexin 43,which is capable of being induced by carotenoids, is the most widelyexpressed connexin in human tissues. Carotenoids, and carotenoidstructural analogs, therefore, may be used for the treatment ofarrhythmia.

Prevention of Cancer: Pharmacologic Agents Used in Previous AnimalTrials

Carotenoids have been evaluated, mostly in animal models, for theirpossible therapeutic value in the prevention and treatment of cancer.Previously the antioxidant properties of carotenoids were the focus ofstudies directed towards carotenoids and their use in cancer prevention.Studies conducted by Bertram et al. (1991) pointed towards the fact thatalthough carotenoids were antioxidants, this particular property did notappear to be the major factor responsible for their activity as cancerchemopreventive agents. It was, however, discovered that the activity ofcarotenoids was strongly correlated with their ability to upregulate gapjunctional communication. It has been postulated that gap junctionsserve as conduits for antiproliferative signals generated bygrowth-inhibited normal cells. Connexin 43, which is capable of beinginduced by carotenoids, is the most widely expressed connexin in humantissues. Upregulation of connexin 43, therefore, may be the mechanism bywhich carotenoids are useful in the chemoprevention of cancer in humansand other animals. And recently, a human study by Nishino et al. (2003)demonstrated that a cocktail of carotenoids (10 mg lycopene, 5 mg eachof α- and β-carotene) given by chronic oral administration wasefficacious in the chemoprevention of hepatocellular carcinoma inhigh-risk cirrhotic patients in Japan. It is likely, then, that morepotent cancer-chemopreventive carotenoids (such as astaxanthin), whichaccumulate more dramatically in liver, will be particularly usefulembodiments.

Use of Carotenoids for the Treatment of Ischemia-Reperfusion Injury,Liver Disease, Arrhythmia, and Cancer

As used herein the terms “inhibiting” and “ameliorating” are generallydefined as the prevention and/or reduction of the negative consequencesof a disease state. Thus, the methods and compositions described hereinmay have value as both an acute and a chronic (prophylactic) modality.

As used herein the term “ischemia-reperfusion injury” is generallydefined as the pathology attributed to reoxygenation of previouslyischemic tissue (either chronically or acutely ischemic), which includesatherosclerotic and thromboembolic vascular disease and its relatedillnesses. In particular, major diseases or processes includingmyocardial infarction, stroke, peripheral vascular disease, venous orarterial occlusion, organ transplantation, coronary artery bypass graftsurgery, percutaneous transluminal coronary angioplasty, andcardiovascular arrest and/or death are included, but are not seen aslimiting for other pathological processes which involve reperfusion ofischemic tissue in their individual pathologies.

As used herein the term “arrhythmia” is generally defined as anyvariation from the normal rhythm of the heart beat, including sinusarrhythmia, premature beat, heart block, atrial fibrillation, atrialflutter, ventricular tachycardia, ventricular fibrillation, pulsusalternans and paroxysmal tachycardia. As used herein the term “cardiacarrhythmia” is generally defined as a disturbance of the electricalactivity of the heart that manifests as an abnormality in heart rate orheart rhythm. Arrhythmia is most commonly related to cardiovasculardisease, and in particular, ischemic heart disease.

As used herein the term “cancer” is generally considered to becharacterized by the uncontrolled, abnormal growth of cells. Inparticular, cancer may refer to tissue in a diseased state includingcarcinogen-initiated and carcinogen-transformed cells.

As used herein the terms “structural carotenoid analogs” may begenerally defined as carotenoids and the biologically active structuralanalogs thereof. Typical analogs include molecules which demonstrateequivalent or improved biologically useful and relevant function, butwhich differ structurally from the parent compounds. Parent carotenoidsare selected from the more than 600 naturally-occurring carotenoidsdescribed in the literature, and their stereo- and geometric isomers.Such analogs may include, but are not limited to, esters, ethers,carbonates, amides, carbamates, phosphate esters and ethers, sulfates,glycoside ethers, with or without spacers (linkers).

As used herein the terms “the synergistic combination of more than onestructural analog of carotenoids” may be generally defined as anycomposition including one structural carotenoid analog combined with oneor more other structural carotenoid analogs or co-antioxidants, eitheras derivatives or in solutions and/or formulations.

As used herein the terms “subject” may be generally defined as allmammals, in particular humans.

As used herein the terms “administration” may be generally defined asthe administration of the pharmaceutical or over-the-counter (OTC) ornutraceutical compositions by any means that achieves their intendedpurpose. For example, administration may include parenteral,subcutaneous, intravenous, intracoronary, rectal, intramuscular,intra-peritoneal, transdermal, or buccal routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, weight, and diseasestate of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired. Any techniquesdescribed herein directed towards the inhibition of ischemia-reperfusioninjury may also be applied to the inhibition or amelioration of a liverdisease, a non-limiting example being Hepatitis C infection. Techniquesdescribed herein directed towards the inhibition and/or amelioration ofischemia-reperfusion injury may also be applied to the inhibition and/oramelioration of arrhythmia. Techniques described herein directed towardsthe inhibition and/or amelioration of ischemia-reperfusion injury mayalso be applied to the inhibition and/or amelioration of cancer.

An embodiment may include the administration of structural carotenoidanalogs alone or in combination to a subject such that the occurrence ofischemia-reperfusion injury is thereby inhibited and/or ameliorated. Thestructural carotenoid analogs may be water soluble and/or waterdispersible derivatives. The carotenoid derivatives may include anysubstituent that substantially increases the water solubility of thenaturally occurring carotenoid. The carotenoid derivatives may retainand/or improve the antioxidant properties of the parent carotenoid. Thecarotenoid derivatives may retain the non-toxic properties of the parentcarotenoid. The carotenoid derivatives may have increasedbioavailability, relative to the parent carotenoid, upon administrationto a subject. The parent carotenoid may be naturally occurring.

Another embodiment may include the administration of a compositioncomprised of the synergistic combination of more than one structuralanalog of carotenoids to a subject such that the occurrence ofischemia-reperfusion injury is thereby reduced. The composition may be a“racemic” (i.e. mixture of the potential stereoisomeric forms) mixtureof carotenoid derivatives. Included as well are pharmaceuticalcompositions comprised of structural analogs of carotenoids incombination with a pharmaceutically acceptable carrier (e.g., humanserum albumin). In one embodiment, structural analogs of carotenoids maybe complexed with human serum albumin (i.e., HSA) in a solvent. HSA mayact as a pharmaceutically acceptable carrier.

In some embodiments, compositions may include all compositions of 1.0gram or less of a particular structural carotenoid analog, incombination with 1.0 gram or less of one or more other structuralcarotenoid analogs and/or co-antioxidants, in an amount which iseffective to achieve its intended purpose. While individual subjectneeds vary, determination of optimal ranges of effective amounts of eachcomponent is with the skill of the art. Typically, a structuralcarotenoid analog may be administered to mammals, in particular humans,orally at a dose of 5 to 100 mg per day referenced to the body weight ofthe mammal or human being treated for ischemia-reperfusion injury.Typically, a structural carotenoid analog may be administered tomammals, in particular humans, parenterally at a dose of between 5 to500 mg per day referenced to the body weight of the mammal or humanbeing treated for reperfusion injury. In other embodiments, about 100 mgof a structural carotenoid analog is either orally or parenterallyadministered to treat or prevent ischemia-reperfusion injury.

The unit oral dose may comprise from about 0.25 mg to about 1.0 gram, orabout 5 to 25 mg, of a structural carotenoid analog. The unit parenteraldose may include from about 25 mg to 1.0 gram, or between 25 mg and 500mg, of a structural carotenoid analog. The unit intracoronary dose mayinclude from about 25 mg to 1.0 gram, or between 25 mg and 100 mg, of astructural carotenoid analog. The unit doses may be administered one ormore times daily, on alternate days, in loading dose or bolus form, ortitrated in a parenteral solution to commonly accepted or novelbiochemical surrogate marker(s) or clinical endpoints as is with theskill of the art.

In addition to administering a structural carotenoid analog as a rawchemical, the compounds may be administered as part of a pharmaceuticalpreparation containing suitable pharmaceutically acceptable carriers,preservatives, excipients and auxiliaries which facilitate processing ofthe structural carotenoid analog which may be used pharmaceutically. Thepreparations, particularly those preparations which may be administeredorally and which may be used for the preferred type of administration,such as tablets, softgels, lozenges, dragees, and capsules, and alsopreparations which may be administered rectally, such as suppositories,as well as suitable solutions for administration by injection or orally,may be prepared in dose ranges that provide similar bioavailability asdescribed above, together with the excipient.

The pharmaceutical preparations may be manufactured in a manner which isitself known to one skilled in the art, for example, by means ofconventional mixing, granulating, dragee-making, softgel encapsulation,dissolving, extracting, or lyophilizing processes. Thus, pharmaceuticalpreparations for oral use may be obtained by combining the activecompounds with solid and semi-solid excipients and suitablepreservatives, and/or co-antioxidants. Optionally, the resulting mixturemay be ground and processed. The resulting mixture of granules may beused, after adding suitable auxiliaries, if desired or necessary, toobtain tablets, softgels, lozenges, capsules, or dragee cores.

Suitable excipients may be fillers such as saccharides (e.g., lactose,sucrose, or mannose), sugar alcohols (e.g., mannitol or sorbitol),cellulose preparations and/or calcium phosphates (e.g., tricalciumphosphate or calcium hydrogen phosphate). In addition binders may beused such as starch paste (e.g., maize or corn starch, wheat starch,rice starch, potato starch, gelatin, tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone). Disintegrating agents may be added (e.g., theabove-mentioned starches) as well as carboxymethyl-starch, cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof (e.g.,sodium alginate). Auxiliaries are, above all, flow-regulating agents andlubricants (e.g., silica, talc, stearic acid or salts thereof, such asmagnesium stearate or calcium stearate, and/or polyethylene glycol, orPEG). Dragee cores are provided with suitable coatings which, ifdesired, are resistant to gastric juices. Softgelatin capsules(“softgels”) are provided with suitable coatings, which, typically,contain gelatin and/or suitable edible dye(s). Animal component-free andkosher gelatin capsules may be particularly suitable for the embodimentsdescribed herein for wide availability of usage and consumption. Forthis purpose, concentrated saccharide solutions may be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethyleneglycol (PEG) and/or titanium dioxide, lacquer solutions and suitableorganic solvents or solvent mixtures, including dimethylsulfoxide(DMSO), tetrahydrofuran (THF), acetone, ethanol, or other suitablesolvents and co-solvents. In order to produce coatings resistant togastric juices, solutions of suitable cellulose preparations such asacetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate,may be used. Dye stuffs or pigments may be added to the tablets ordragee coatings or softgelatin capsules, for example, for identificationor in order to characterize combinations of active compound doses, or todisguise the capsule contents for usage in clinical or other studies.

Other pharmaceutical preparations which may be used orally includepush-fit capsules made of gelatin, as well as soft, thermally-sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules may contain the active compounds in the form ofgranules which may be mixed with fillers such as, for example, lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers and/or preservatives. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils such as rice bran oil or peanut oil or palmoil, or liquid paraffin. In other embodiments, stabilizers andpreservatives may be added.

Possible pharmaceutical preparations which may be used rectally include,for example, suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, or parrafin hydrocarbons.In addition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the active compounds with a base. Possiblebase materials include, for example, liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include, but are notlimited to, aqueous solutions of the active compounds in water-solubleand/or water dispersible form, for example, water-soluble salts, esters,carbonates, phosphate esters or ethers, sulfates, glycoside ethers,together with spacers and/or linkers. In addition, suspensions of theactive compounds as appropriate oily injection suspensions may beadministered, particularly suitable for intramuscular injection.Suitable lipophilic solvents, co-solvents (such as DMSO or ethanol), orvehicles including fatty oils, for example, rice bran oil or peanut oiland/or palm oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides, may be used. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspensionincluding, for example, sodium carboxymethyl cellulose, sorbitol,dextran, and/or cyclodextrins. Cyclodextrins (e.g., β-cyclodextrin) maybe used specifically to increase the water solubility for parenteralinjection of the structural carotenoid analog. Liposomal formulations,in which mixtures of the structural carotenoid analog with, for example,egg yolk phosphotidylcholine (E-PC), may be made for injection.Optionally, the suspension may also contain stabilizers, for example,antioxidants such as BHT, or preservatives, such as benzyl alcohol.

EXAMPLES

Having now described the invention, the same will be more readilyunderstood through reference to the following example(s), which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

Regarding the synthesis and characterization of compounds describedherein, reagents were purchased from commercial sources and used asreceived unless otherwise indicated. Solvents for reactions andisolations were reagent grade and used without purification unlessotherwise indicated. All of the following reactions were performed undernitrogen (N₂) atmosphere and were protected from direct light. “Racemic”astaxanthin (as the mixture of stereoisomers 3S,3′S, meso, and 3R,3′R ina 1:2:1 ratio) was purchased from Divi's Laboratories, Ltd (BucktonScott, India). “Racemic” lutein and zeaxanthin were purchased fromIndofine Chemical Co., Inc. Thin-layer chromatography (TLC) wasperformed on Uniplate Silica gel GF 250 micron plates. HPLC analysis forin-process control (IPC) was performed on a Varian Prostar Series 210liquid chromatograph with an Alltech Rocket, Platinum-C18, 100 Å, 3 μm,7×53 mm, PN 50523; Temperature: 25° C.; Mobile phase: (A=water; B=10%dichloromethane/methanol), 40% A/60% B (start); linear gradient to 100%B over 8 min; hold 100% B over 4 min linear gradient to 40% A/60% B over1 min; Flow rate: 2.5 mL/min; Starting pressure: 2050 PSI; PDA Detectorwavelength: 474 nm. NMR was recorded on a Bruker Advance 300 and massspectroscopy was taken on a ThermoFinnigan AQA spectromometer. LC/MS wasrecorded on an Agilent 1100 LC/MSD VL ESI system; column: Zorbax EclipseXDB-C18 Rapid Resolution (4.6×75 mm, 3.5 μm, USUT002736); temperature:25° C.; starting pressure: 107 bar; flow rate: 1.0 mL/min.; mobile phase(% A=0.025% TFA in H₂O, % B=0.025% TFA in acetonitrile) Method 1(compounds 8-21, 23-27, 30,31): 70% A/30% B (start), step gradient to50% B over 5 min.; step gradient to 98% B over 8.30 min., hold at 98% Bover 15.20 min., step gradient to 30% B over 15.40 min.; Method 2(compounds 28,29): 70% A/30% B (start), step gradient to 50% B over 4min., step gradient to 90% B over 7.30 min., step gradient to 98% B over10.30 min., hold at 98% B over 15.20 min., step gradient to 30% B over15.40 min.; Method 3 (compound 22): 70% A/30% B (start), step gradientto 50% B over 5 min., step gradient to 98% B over 8.30 min., hold at 98%B over 25.20 min., step gradient to 30% B over 25.40 min.; PDA Detector:470 nm; LRMS: +mode, ESI.

Astaxanthin (2E). HPLC retention time: 11.629 min., 91.02% (AUC); LRMS(ESI) m/z (relative intensity): 598 (M⁺+2H) (60), 597 (M⁺+H) (100); HPLCretention time: 12.601 min., 3.67% (AUC); LRMS (ESI) m/z (relativeintensity): 597 (M⁺+H) (100); HPLC retention time: 12.822 min., 5.31%(AUC); LRMS (ESI) m/z (relative intensity): 597 (M⁺+H) (100).

Lutein (XXX). HPLC retention time: 12.606 min., 100% (AUC); LRMS (ESI)m/z (relative intensity): 568 (M⁺) (100).

Zeaxanthin (XXXI). HPLC retention time: 12.741 min., 100% (AUC); LRMS(ESI) m/z (relative intensity): 568 (M⁺) (100).

Example 1 Synthesis of XV (the Disuccinic Acid ester of Astaxanthin(Succinic acidmono-(4-{18-[4-(3-carboxy-propionyloxy)-2,6,6-trimethyl-3-oxo-cyclohex-1-enyl]-3,7,12,16-tetramethyl-octadeca-1,3,5,7,9,11,13,15,17-nonaenyl}-3,5,5-trimethyl-2-oxo-cyclohex-3-enyl)ester))

To a solution of astaxanthin 2E (6.0 g, 10.05 mmol) in DCM(“dichloromethane”) (50 mL) at room temperature was added DIPEA(“N,N-diisopropylethylamine”) (35.012 mL, 201 mmol), succinic anhydride(10.057 g, 100.5 mmol), and DMAP (“4-(dimethylamino)pyridine”) (0.6145g, 5.03 mmol). The reaction mixture was stirred at room temperature for48 hours, at which time the reaction was diluted with DCM, quenched withbrine/1M HCl (60 mL/10 mL), and then extracted with DCM. The combinedorganic layers were dried over Na₂SO₄ and concentrated to yieldastaxanthin disuccinate (XV) (100%) HPLC retention time: 10.031 min.,82.57% (AUC); LRMS (ESI) m/z (relative intensity): 798 (M⁺+2H) (52), 797(M⁺+H) (100); HPLC retention time: 10.595 min., 4.14% (AUC); LRMS (ESI)m/z (relative intensity): 797 (M⁺+H) (40), 697 (100); HPLC retentiontime: 10.966 min., 5.68% (AUC); LRMS (ESI) m/z (relative intensity): 797(M⁺+H) (100), 679 (31); HPLC retention time: 11.163 min., 7.61% (AUC);LRMS (ESI) m/z (relative intensity): 797 (M⁺+H) (38), 679 (100), and nodetectable astaxanthin 2E.

Example 2 Synthesis of XVI (the Disodium Salt of the Disuccinic Acidester of Astaxanthin (Succinic acidmono-(4-{18-[4-(3-carboxy-propionyloxy)-2,6,6-trimethyl-3-oxo-cyclohex-1-enyl]-3,7,12,16-tetramethyl-octadeca-1,3,5,7,9,11,13,15,17-nonaenyl}-3,5,5-trimethyl-2-oxo-cyclohex-3-enyl)ester))

Disuccinic acid ester of astaxanthin (2 g, 2.509 mmol) and 200 mLethanol was stirred at room temperature under nitrogen in a 500 mLround-bottom flask. Sodium ethoxide (340 mg, 5.019 mmol, Acros#A012556101) was added as a solid in a single portion and the solutionwas allowed to stir overnight. The following day, the precipitate wasfiltered off and washed with ethanol followed by methylene chloride toafford a purple solid, the disodium salt of the disuccinic acid ester ofastaxanthin, XVI [1.41 g, 67%] and was placed on a high vacuum line todry. ¹H-NMR (Methanol-d₄) δ 6.77-6.28 (14 H, m), 5.53 (2 H, dd, J=12.6,6.8), 2.68-2.47 (8 H, m), 2.08-1.88 (22 H, m), 1.37 (6 H, s), 1.24 (6H,s); ¹³C NMR (CDCl₃) δ 196.66, 180.80, 175.01, 163.69, 144.12, 141.38,138.27, 136.85, 136.12, 135.43, 132.35, 129.45, 126.22, 124.71, 72.68,44.09, 38.63, 34.02, 32.34, 31.19, 26.86, 14.06, 13.19, 12.91; Massspectroscopy +ESI, 819.43 monosodium salt, 797.62 disuccinic acid esterof astaxanthin; HPLC 7.41 min (99.84%).

Example 3 Synthesis of the BocLys(Boc)OH ester of Astaxanthin (XXI)

HPLC: Column: Waters Symmetry C18 3.5 micron 4.6 mm×150 mm; Temperature:25° C.; Mobile phase: (A=0.025% TFA in H₂O; B=0.025% TFA in MeCN), 95%A/5% B (start); linear gradient to 100% B over 12 min, hold for 4 min;linear gradient to 95% B/5% A over 2 min; linear gradient to 95% A/5% Bover 4 min; Flow rate: 2.5 mL/min; Detector wavelength: 474 nm.

To a mixture of astaxanthin 2E (11.5 g, 19.3 mmol) and BocLys(Boc)OH(20.0 g, 57.7 mmol) in methylene chloride (500 mL) were added4-dimethylaminopyridine (DMAP) (10.6 g, 86.6 mmol) and1,3-diisopropylcarbodiimide (“DIC”) (13.4 g, 86.7 mmol). Theround-bottomed flask was covered with aluminum foil and the mixture wasstirred at ambient temperature under nitrogen overnight. After 16 hours,the reaction was incomplete by HPLC and TLC. An additional 1.5equivalents of DMAP and DIC were added to the reaction and after 2hours, the reaction was complete by HPLC. The mixture was thenconcentrated to 100 mL and a white solid (1,3-diisopropylurea) wasfiltered off. The filtrate was flash chromatographed through silica gel(10% to 50% Heptane/EtOAc) to give the desired product as a dark redsolid (XXI) (28.2 g, >100% yield). ¹H NMR (DMSO-d₆) δ 7.24 (2 H, t,J=6.3 Hz), 6.78 (2 H, d, 5.0 Hz), 6.57-6.27 (14 H, m), 5.50-5.41 (2 H,m), 3.99-3.97 (2 H, d, 6.0 Hz), 2.90 (4 H, m), 2.03 (4 H, m), 2.00 (6 H,s), 1.97 (6 H, s), 1.82 (6 H, s), 1.70-1.55 (4 H, m), 1.39-1.33 (36 H,m), 1.24-1.13 (8 H, m), 1.01-0.99 (6 H, m), 0.86-0.83 (6 H, m). HPLC:21.3 min (24.6% AUC)); 22.0 min (48.1% (AUC)); 22.8 min (20.6% (AUC)).TLC (1:1 Heptane/EtOAc: R_(f)0.41; R_(f)0.5; R_(f)0.56). LC/MS analysiswas performed on a Agilent 1100 LC/MSD VL ESI system by flow injectionin positive mode; Mobile Phase: A=0.025% TFA in H₂O; B=0.025% TFA inMeCN, 10% A/90% B(start); Starting pressure: 10 bar; PDA Detector 470nm. +ESI, m/z=1276.1(M+Na⁺).

Example 4 Synthesis of the Tetrahydrochloride Salt of the Dilysinateester of Astaxanthin (XX)

A mixture of DiBocLys(Boc) ester of astaxanthin (XXI) (20.0 g, 16.0mmol) and HCl in 1,4-dioxane (4.00 M, 400 mL, 1.60 mol, 100 eq) wasstirred at ambient temperature under a nitrogen atmosphere. Theround-bottomed flask was covered with aluminum foil and the reaction wasstirred for 1 hour, at which time the reaction was complete by HPLC. Thetitle compound precipitated and was collected by filtration, washed withether (3×100 mL) and dried (14.7 g, 92%, 91.6% purity by HPLC). Aportion (13.5 g) of the crude solid was dissolved in 500 mL of a 1:2methanol/methylene chloride mixture and stirred under nitrogen. Diethylether (168 mL) was then added dropwise and the precipitated solid wascollected by filtration to afford the desired product as a dark redsolid (8.60 g, 63.7% yield). ¹H NMR (DMSO-d₆) δ 8.65 (6 H, s), 8.02 (6H, s), 6.78-6.30 (14 H, m), 5.59-5.51 (2 H, m), 4.08 (2 H, m), 2.77 (4H, m), 2.09-2.07 (4 H, m), 2.01 (6 H, s), 1.97 (6 H, s), 1.90-1.86 (4 H,m), 1.84 (6 H, m) 1.61-1.58 (8 H, m), 1.37 (6 H, s), 1.22 (6 H, s).HPLC: 7.8 min (97.0% (AUC)). LC/MS analysis was performed on an Agilent1100 LC/MSD VL ESI system with Zorbax Eclipse XDB-C18 Rapid Resolution4.6×75mm, 3.5 microns, USUT002736; Temperature: 25° C.; Mobile Phase: (%A=0.025% TFA in H₂O; % B=0.025% TFA in MeCN), 70% A/30% B (start);linear gradient to 50% B over 5 min, linear gradient to 100% B over 7min; Flow rate: 1.0 mL/min; Starting pressure: 108 bar; PDA Detector 470nm. Mass spectrometry +ESI, m/z=853.9(M+H⁺), m/z=875.8(M+Na⁺); LC 4.5min.

Example 5 Synthesis of the Bis-(2-OTBS Ascorbic Acid) 6-Ester ofAstaxanthin Disuccinate (XXII)

HPLC: Column: Waters Symmetry C18 3.5 micron 4.6 mm×150 mm; Temperature:25° C.; Mobile phase: (A=0.025% TFA in water; B=0.025% TFA inacetonitrile), 95% A/5% B (start); linear gradient to 100% B over 5 min,hold for 10 min; linear gradient to 95% B over 2 min; linear gradient to95% A/5% B over 3 min; Flow rate: 1.0 mL/min; Detector wavelength: 474nm.

To a stirring solution of astaxanthin disuccinate (XV) (20.00 g, 25.1mmol) in 600 mL of dichloromethane was added 4-dirnethylaminopyridine(DMAP) (6.13 g, 50.2 mmol), 2-O-tert-butyldimethylsilyl (OTBS) ascorbicacid (XXVI) (21.86 g, 75.3 mmol), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI-HCl)(12.02 g, 62.75 mmol). After 14 h, the reaction mixture was flashchromatographed through silica gel (1.0 kg silica gel, eluent 0.5%HOAc/5% MeOH/EtOAc). Fraction 10 was concentrated to afford dark redsolid (6.47 g, 19.2% yield, 58% AUC purity by HPLC). The crude productwas flashed chromatographed through silica gel (600 g silica gel, eluent0.25% HOAc/5% MeOH/EtOAc). Fractions 6-10 were concentrated under vacuumto afford dark red solid (1.50 g, 4.4% yield, 94.8% AUC purity by HPLC¹H-NMR (CDCl₃) δ 11.13 (2H, s), 6.78-6.28 (14 H, m), 5.43 (2 H, dd,J=12.2, 7.1 Hz), 5.34 (2H, s), 4.78 (2H, d, J=5.4 Hz), 4.11-4.07 (6H,m), 2.69-2.65 (8 H, m), 2.05-1.97 (22 H, m), 1.81 (6 H, s), 1.33 (6 H,s), 0.92 (18 H, s), 0.15 (6 H, s), 0.14 (6H, s); HPLC 13.4 min [94.8%(AUC)]; Mass spectroscopy −ESI, m/z=1340.6 (M⁻).

Example 6 Synthesis of the Bis-Ascorbic Acid 6-Ester of AstaxanthinDisuccinate (XIX)

To a stirring solution of the bis-(2-OTBS ascorbic acid) 6-ester ofastaxanthin disuccinate (XXII) (100 mg, 0.075 mmol) in THF (5 mL) at 0°C. was added HF.Et₃N (121 μL, 0.745 mmol). The reaction was stirred fpr1 h at 0° C. then warmed to rt. The reaction was stirred 2.5 h beforebeing quenched by pouring into a separatory funnel containing 5 mL IPACand 5 mL of water. The aqueous layer was removed and the organic layerwas washing with water (2×5 mL). The organic solvents were removed byrotary evaporation to give a dark red solid, which was used withoutpurification. ¹H-NMR (CDCl₃) δ 11.12 (2 H, s), 8.40 (2 H, s), 6.87-6.28(14 H, m) 5.43-5.32 (4 H, m), 4.69 (s, 2H), 4.09 (s, 4H), 3.99 (s, 2H),2.68-2.50 (m, 8H), 2.00-1.76 (22 H, m), 1.36-1.19 (12 H, m); HPLC 8.9min [80.7% (AUC)]; Mass spectroscopy +ESI, m/z=1113.2 (M+H⁺).

Example 7 Synthesis of the Sodium Salt of the Bis-Ascorbic Acid 6-Esterof Astaxanthin Disuccinate (XXIII)

To a stirring solution of the crude bis-ascorbic acid 6-ester ofastaxanthin disuccinate (XIX) (0.075 mmol) in acetone (5 mL) at rt wasadded triethylorthoformate (62 μL, 0.373 mmol). The solution was stirred15 min then a solution of sodium 2-ethylhexanoate in acetone (93 μL,0.019 mmol, 0.20 M) was added dropwise. The resulting precipitate wasremoved by filtration. The filtrate was cooled to 0° C. and treated withadditional sodium 2-ethylhexanoate in acetone (373 μL, 0.075 mmol, 0.20M). The reaction was stirred for 5 min then the solid material wascollected by filtration, washed with acetone (5 mL), and dried underhigh vacuum to give a dark red solid (27.8 mg, 32.2% yield): HPLC 8.9min [88.2% (AUC)], Mass spectroscopy +APCI, m/z=1113.3 (M+3H-2Na⁺).

Example 8 Synthesis of the Dicyclohexylmethyl Ester of AstaxanthinDisuccinate (XXIV)

HPLC: Column: Alltech Rocket, Platinum-C18, 100 Å, 3 μm, 7×53 mm;Temperature: 25° C.; Mobile phase: (A=0.025% TFA in water; B=0.025% TFAin acetonitrile), 70% A/30% B (start); hold for 40 sec; linear gradientto 50% B over 4 min 20 sec; linear gradient to 100% B over 1 min 30 sec,hold for 4 min 40 sec; linear gradient to 70% A/30% B in 20 sec; Flowrate: 2.5 mL/min; Detector wavelength: 474 nm.

To a stirred solution of the astaxanthin disuccinate (XV) (100 mg, 0.125mmol) and N,N-dimethylformamide (6.0 mL) in a 25 mL round-bottom flaskwas added cesium carbonate (90.0 mg, 0.275 mmol) at room temperatureunder N₂ and covered with aluminum foil. The reaction was stirred for 15minutes then bromomethyl cyclohexane (52.0 μL, 0.375 mmol) was added.After 2 days, the reaction was quenched by adding 4 mL of a saturatedsolution of sodium bicarbonate and diluted with 50 mL ofdichloromethane. The diluted solution was washed twice with 25 mL ofwater before drying over anhydrous sodium sulfate. The organic solutionwas filtered and the solvent was removed by rotary evaporation. Thecrude residue was purified by flash chromatography (10-50%EtOAc/heptane) to afford a dark red solid (40.2 mg, 32.5% yield): ¹H-NMR(CDCl₃) δ 7.03-6.17 (14 H, m), 5.54 (2 H, dd, J=12.9, 6.7 Hz), 3.92 (4H, d, J=6.4 Hz), 2.82-2.63 (8 H, m), 2.08-1.92 (14 H, m), 1.90 (6 H, s),1.75-1.62 (14 H, m), 1.34-1.20 (22 H, m); HPLC 8.9 min [83.9% (AUC)];TLC (3:7 EtOAc/heptane: R_(f)0.38); Mass spectroscopy +ESI, m/z=989.6(M+H⁺).

Example 9 Synthesis of 2-OTBS-5,6-Isopropyledine Ascorbic Acid (XXV)

HPLC: Alltech Rocket, Platinum-C18, 100A, 3 μm, 7×53 mm, PN 50523;Temperature: 25° C.; Mobile phase: (A=0.025% TFA in water; B=0.025% TFAin acetonitrile), 90% A/10% B (start); linear gradient to 30% B over 3min; linear gradient to 90% B over 3 min, hold for 2 min; lineargradient to 90% A/10% B over 1 min, then hold for 1 min; Flow rate: 2.5mL/min; Detector wavelength: 256 nm.

To a stirring solution of 5,6-isopropyledine ascorbic acid (100.0 g, 463mmol) in 1.00 L THF was added tert-butyldimethylsilyl chloride (TBSCI)(76.7 g, 509 mmol) at rt followed by addition ofN,N-diisopropylethylamine (DIPEA) (161 mL, 925 mmol) over 30 min. Thereaction was stirred 14 h at rt, then concentrated under vacuum. Themixture was dissolved in methyl tert-butyl ether (MTBE) (1.00 L) andextracted with 1 M potassium carbonate (1.00 L) in a separatory funnel.The aqueous layer was extracted one more time with MTBE (1.00 L), andthe pH of the aqueous layer was adjusted to pH 6 using 2 N HCl. Theaqueous layer was extracted twice with isopropyl acetate (IPAC) (1.00 L)and concentrated to afford an off white solid (150.4 g, 98% yield):¹H-NMR (DMSO d₆) δ 11.3 (1 H, s), 4.78 (1 H, d, J=2.0 Hz), 4.41-4.36 (1H, dd, J=8.4, 7.4 Hz), 3.92 (1 H, dd, J=8.4, 6.0), 1.24 (3 H, s), 1.23(3 H, s), 0.92 (9 H, s), 0.14 (6H, s); HPLC 5.9 min [91.6% (AUC)]; Massspectroscopy −ESI, m/z=329.2 (M−H⁻).

Example 10 Synthesis of 2-OTBS Ascorbic Acid (XXVI)

To a stirring solution of 2-OTBS-5,6-isopropyledine ascorbic acid (XXV)(150.4 g, 455 mmol) in 1.50 L of dichloromethane at rt was addedpropanedithiol (54.0 mL, 546 mmol) under nitrogen. The solution wascooled to −45° C., and then BF₃—OEt₂ (58.0 mL, 455 mmol) was addeddropwise at a rate that kept the temperature below −40° C. After 1 h,the reaction was complete by HPLC. The reaction was quenched by pouringthe cold reaction mixture into a separatory funnel containing 1.00 L ofIPAC and 500 mL of a saturated solution of ammonium chloride and 500 mLof water. The organic layer was concentrated to a white solid. In orderto purge the propane dithiol, the solid was reslurried indichloromethane (250 mL) for 2 h and heptane (1.00 L) was added andstirred for 1 h. The mixture was concentrated under vacuum to a volumeof 500 mL. The mixture was filtered and dried under vacuum to afford anof white solid (112.0 g, 85% yield): ¹H-NMR (DMSO d₆) δ 11.0 (1 H, s),4.89 (2 H, s), 4.78 (1 H,d, J=1.2 Hz), 3.82-3.80 (1 H, m), 3.45-3.42 (2H, m), 0.923 (9 H, s), 0.14 (6H, s); HPLC 4.9 min [92.0% (AUC)]; Massspectroscopy −ESI, m/z=289.0 (M−H⁻).

Example 11 Synthesis of the bis-dimethylphosphate Ester of Astaxanthin(XXVI)

HPLC: Waters Symmetry C18, 3 μm, 4.6×150 mm, WAT200632, Temperature: 25°C.; Mobile phase: (A=water; B 10% DCM/MeOH), 10% A/90% B (start); lineargradient to 100% B over 9 min; hold 100% B over 11 min, linear gradientto 10% A/90% B over 1 min; Flow rate: 1.0 mL/min; Detector wavelength:474 nm.

To a mixture of astaxanthin 2E (500 mg, 0.84 mmol) and methyl imidazole(0.50 mL, 6.27 mmol) in methylene chloride at 37° C. was addeddimethylbromophosphate (2 M, 5.04 mL) (Ding, 2000). After 24 h, thereaction was not complete by HPLC and dimethylbromophosphate (2 M, 5.04mL) was added. After 48 h, the reaction was not complete by HPLC anddimethylbromophosphate (2 M, 5.04 nmL) was added. After 72 h, thereaction was complete by HPLC. The reaction was diluted with methylenechloride (20 mL) and quenched with water (20 mL). The layers wereseparated and the aqueous layer was extracted again with 20 mL ofmethylene chloride. The organic layers were combined and concentratedunder vacuum to afford 2.69 g (>100% yield). ¹H NMR (CDCl₃) δ 6.58-6.14(14 H, m), 5.05-4.95 (2 H, m), 3.91-3.60 (12 H, m), 2.11-2.04 (4 H, m),2.04-1.92 (12 H, m), 1.85 (6 H, s), 1.26 (6 H, s), 1.15 (6 H, s). HPLC:4.29 min (86.7% AUC)). Mobile Phase: A=0.025% TFA in H₂O; B=0.025% TFAin acetonitrile, 10% A/90% B(start); PDA Detector 474 nm. +ESI,m/z=813.62 (M+1).

Example 12 Synthesis of the BocProOH ester of Astaxanthin (XXVIII)

LC/MS Analysis: LC/MS analysis was performed on an Agilent 1100 LC/MSDVL ESI system with Zorbax Eclipse XDB-C18 Rapid Resolution 4.6×75 mm,3.5 μm, USUT002736; Temperature: 25° C.; Mobile Phase:(% A=0.025% TFA inH₂O; % B=0.025% TFA in MeCN), 70% A/30% B(start); linear gradient to 50%B over 5 min, linear gradient to 98% B over 3 min, hold at 98% B for 17min; Flow rate: 1.0 mL/min; Starting pressure: 108 bar; PDA Detector 470nm, 373 nm, 214 nm. LRMS: +mode, ESI.

To a mixture of astaxanthin 2E (5.00 g, 8.38 mmol) and BocProOH (10.8 g,50.3 mmol) in methylene chloride (500 mL) were added4-dimethylaminopyridine (DMAP) (6.14 g, 50.3 mmol) and1,3-diisopropylcarbodimide (DIC) (7.79 mL, 50.3 mmol). The mixture wasstirred at ambient temperature under nitrogen overnight. After 16 hours,the reaction was complete by TLC. The mixture was then concentrated todryness and the crude residue was slurried with 100 mL of diethyl etherand filtered through a pad of Celite. The filtrate was flashchromatographed through silica gel (Et₂O) to give the desired product asa dark red solid (8.56 g, >100% yield). LC: 17.5 min [23.1% AUC)]; 18.2min [45.1% (AUC)]; 19.4 min [22.0% (AUC)]TLC (3:2 EtOAc/Hexane:R_(f)0.51; R_(f)0.55; R_(f)0.59). MS+ESI, m/z=1013.8 (M+Na⁺).

Example 13 Synthesis of the Dihydrochloride Salt of the Diprolinateester of Astaxanthin (XXIX)

A mixture of diethyl ether (130 mL) and EtOH (48.9 mL, 838 mmol) wascooled to −78° C. under a nitrogen atmosphere. Acetyl chloride (82.0 mL,838 mmol) was added dropwise to the cooled mixture over 30 minutes. Thereaction was removed from the cooling bath and allowed to slowly warm toroom temperature. The contents of the flask were poured into a separateround-bottomed flask containing DiBocPro ester of astaxanthin (XXVIII)(8.31 g, 8.38 mmol) and a stirrer bar. The flask was covered withaluminum foil and the reaction was stirred at ambient temperature undernitrogen overnight. After 16 hours the reaction was complete by LC. Thetitle compound precipitated and was collected by filtration, washed withether (3×100 mL) and dried (6.37 g, 88.0% crude yield, 75.2% purity byLC). LC: 8.00 min [75.2% (AUC)]. MS +ESI, m/z=791.7 (M+H⁺).

Example 14 Synthesis of Lutein disuccinate (XXXII)

To a solution of lutein (XXX) (0.010 g, 0.018 mmol) in DCM (2 mL) atroom temperature was added DIPEA (0.063 mL, 0.360 mmol), succinicanhydride (0.036 g, 0.360 mmol), and DMAP (0.021 g, 0.176 mmol). Thereaction mixture was stirred at room temperature for 48 hours, at whichtime the reaction was diluted with DCM, quenched with brine/1M HCl(6mL/1 mL), and then extracted with DCM. The combined organic layerswere dried over Na₂SO₄ and concentrated to yield lutein disuccinate(XXXII) (93.09%) HPLC retention time: 11.765 min., 93.09% (AUC); LRMS(ESI) m/z (relative intensity): 769 (M⁺) (24), 651 (100), and nodetectable lutein.

Example 15 Synthesis of Succinic acid esters of zeaxanthin (XXXIII,XXXIV)

To a solution of zeaxanthin (XXXI) (0.010 g, 0.018 mmol) in DCM (2 mL)at room temperature was added DIPEA (0.063 mL, 0.360 mmol), succinicanhydride (0.036 g, 0.360 mmol), and DMAP (0.021 g, 0.176 mmol). Thereaction mixture was stirred at room temperature for 48 hours, at whichtime the reaction was diluted with DCM, quenched with brine/1M HCl (6mL/1 mL), and then extracted with DCM. The combined organic layers weredried over Na₂SO₄ and concentrated to yield zeaxanthin monosuccinate(XXXIII) (2.86%) HPLC retention time: 12.207 min., 2.86% (AUC); LRMS(ESI) m/z (relative intensity): 669 (M⁺+H) (53), 668 (M⁺) (100),zeaxanthin disuccinate (XXXIV) (97.14%) HPLC retention time: 11.788min., 67.42% (AUC); LRMS (ESI) m/z (relative intensity): 792 (M⁺+Na)(42), 769 (M⁺) (73), 651 (100); HPLC retention time: 13.587 min., 11.19%(AUC); LRMS (ESI) m/z (relative intensity): 792 (M⁺+Na) (36), 769 (M⁺)(38), 663 (100); HPLC retention time: 13.894 min., 18.53% (AUC); LRMS(ESI) m/z (relative intensity): 769 (M⁺) (62), 663 (77), 651 (100), andno detectable zeaxanthin

Example 16 Synthesis of Aconitic acid esters of astaxanthin (XXXV,XXXVI)

To a solution of astaxanthin 2E (0.100 g, 0.168 mmol) in DCM/DMF(“N,N-dimethylformamide”) (4 mL/2 mL) at room temperature was addedDIPEA (0.878 mL, 5.04 mmol), cis-aconitic anhydride (0.2622 g, 1.68mmol), and DMAP (0.4105 g, 3.36 mmol). The reaction mixture was stirredat room temperature for 36 hours, at which time the reaction was dilutedwith DCM, quenched with brine/1M HCl (20 mL/3 mL), and then extractedwith DCM. The combined organic layers were concentrated to yieldaconitic monoester (XXXV) (13.25%) HPLC retention time: 10.485 min.,4.95% (AUC); LRMS (ESI) m/z (relative intensity): 777 (M⁺+Na+2H) (57),623 (100); HPLC retention time: 10.722 min., 8.30% (AUC); LRMS (ESI) m/z(relative intensity): 777 (M⁺+Na+2H) (6), 709 (100), aconitic diester(XXXVI) (27.67%) HPLC retention time: 9.478 min., 15.44% (AUC); LRMS(ESI) m/z (relative intensity): 933 (M⁺+Na+2H) (10), 831 (100); HPLCretention time: 9.730 min., 12.23% (AUC); LRMS (ESI) m/z (relativeintensity): 913 (M⁺+4H) (4), 843 (100), and astaxanthin (44.40%).

Example 17 Synthesis of Citric acid esters of astaxanthin (XXXVI,XXXVIII)

To a suspension of citric acid (0.5149 g, 2.86 mmol) in DCM (8 mL) atroom temperature was added DIPEA (1.167 mL, 0.6.70 mmol), DIC (0.525 mL,3.35 mmol), DMAP (0.4094 g, 3.35 mmol), and astaxanthin (0.200 g, 0.335mmol). The reaction mixture was stirred at room temperature for 36hours, at which time the reaction was diluted with DCM, quenched withbrine/1M HCl (20 mL/3 mL), and then extracted with DCM. The combinedorganic layers were concentrated to yield citric acid monoester (XXXVII)(26.56%) HPLC retention time: 9.786 min., 17.35% (AUC); LRMS (ESI) m/z(relative intensity): 773 (M⁺+3H) (14), 771 (M⁺+H) (100); HPLC retentiontime: 9.989 min., 9.21% (AUC); LRMS (ESI) m/z (relative intensity): 773(M⁺+3H) (50), 771 (M⁺+H) (100), citric acid diester (XXXVIII) (7.81%)HPLC retention time: 8.492 min., 3.11% (AUC); LRMS (ESI) m/z (relativeintensity): 968 (M⁺+Na) (75), 967 (100), 946 (M⁺+H) (37); HPLC retentiontime: 8.708 min., 2.43% (AUC); LRMS (ESI) m/z (relative intensity): 968(M⁺+Na) (95), 946 (M⁺+H) (100); HPLC retention time: 8.952 min., 2.27%(AUC); LRMS (ESI) m/z (relative intensity): 946 (M⁺+H) (19), 500 (100),and astaxanthin (21.26%).

Example 18 Synthesis of Dimethylaminobutyric acid monoester ofastaxanthin (XXXIX)

To a suspension of 4-(dimethylamino)-butyric acid hydrochloride (0.2816g, 1.68 mmol) in DCM/DMF (3 mL/3 mL) at room temperature was added DIPEA(0.878 mL, 5.04 mmol), HOBT (“1-hydroxybenzotriazole”)-H₂O (0.3094 g,2.02 mmol), DMAP (0.4105 g, 3.36 mmol), and astaxanthin (0.100 g, 0.168mmol). The reaction mixture was stirred at room temperature for 36hours, at which time the reaction was diluted with DCM, quenched withbrine/1M HCl (20 mL/3 mL), and then extracted with DCM. The combinedorganic layers were concentrated to yield 4-(dimethylamino)butyric acidmonoester (XXXIX) (24.50%) HPLC retention time: 9.476 min., 20.32%(AUC); LRMS (ESI) m/z (relative intensity): 732 (M⁺+Na) (13), 729 (100);HPLC retention time: 9.725 min., 4.18% (AUC); LRMS (ESI) m/z (relativeintensity): 732 (M⁺+Na) (50), 729 (100), and astaxanthin (61.21%).

Example 19 Synthesis of Glutathione monoester of astaxanthin (L)

To a suspension of reduced glutathione (0.5163 g, 1.68 mmol) in DCM/DMF(3 mL /3 mL) at room temperature was added DIPEA (0.878 mL, 5.04 mmol),HOBT-H₂O (0.3094 g, 2.02 mmol), DMAP (0.4105 g, 3.36 mmol), DIC (0.316mL, 2.02 mmol), and astaxanthin 2E (0.100 g, 0.168 mmol). The reactionmixture was stirred at room temperature for 36 hours, at which time thereaction was diluted with DCM, quenched with brine/1M HCl (20 mL/3 mL),and then extracted with DCM. The combined organic layers wereconcentrated to yield glutathione monoester (L) (23.61%) HPLC retentiontime: 9.488 min., 16.64% (AUC); LRMS (ESI) m/z (relative intensity): 886(M⁺) (13), 810 (54), 766 (100); HPLC retention time: 9.740 min., 3.57%(AUC); LRMS (ESI) m/z (relative intensity): 886 (M⁺) (24), 590 (78), 546(100); HPLC retention time: 9.997 min., 3.40% (AUC); LRMS (ESI) m/z(relative intensity): 886 (M⁺) (25), 869 (85), 507 (100), andastaxanthin (68.17%).

Example 20 Synthesis of Tartaric acid diester of astaxanthin (LI)

To a suspension of (L)-tartaric acid (0.4022 g, 2.68 mmol) in DCM/DMF (5mL/5 mL) at room temperature was added DIPEA (1.167 mL, 0.6.70 mmol),HOBT-H₂O (0.5131 g, 3.35 mmol), DMAP (0.4094 g, 3.35 mmol), andastaxanthin 2E (0.200 g, 0.335 mmol). The reaction mixture was stirredat room temperature for 36 hours, at which time the reaction was dilutedwith DCM, quenched with brine/1M HCl (20 mL/3 mL), and then extractedwith DCM. The combined organic layers were concentrated to yieldtartaric acid diester (LI) (18.44%) HPLC retention time: 9.484 min.,14.33% (AUC); LRMS (ESI) m/z (relative intensity): 884 (M⁺+Na+H) (100),815 (72), 614 (72); HPLC retention time: 9.732 min., 4.11% (AUC); LRMS(ESI) m/z (relative intensity): 883 (M⁺+Na) (100), 539 (72), andastaxanthin (67.11%).

Example 21 Synthesis of Sorbitol monoester of astaxanthin disuccinate(LII)

To a solution of astaxanthin disuccinate (XV) (0.200 g, 0.251 mmol) inDMF (10 mL) at room temperature was added DIPEA (1.312 mL, 7.53 mmol),HOBT-H₂O (0.4610 g, 3.01 mmol), DMAP (0.6133 g, 5.02 mmol), and(D)-sorbitol (0.4572 g, 2.51 mmol). The reaction mixture was stirred atroom temperature for 36 hours, at which time the reaction was dilutedwith DCM, quenched with brine/1M HCl (20 mL/3 mL), and then extractedwith DCM. The combined organic layers were concentrated to yieldsorbitol monoester (LII) (3.52%) HPLC retention time: 9.172 min., 3.52%(AUC); LRMS (ESI) m/z (relative intensity): 984 (M⁺+Na) (28), 503 (100),and astaxanthin disuccinate (91.15%).

Example 22 Synthesis of Sorbitol diester of astaxanthin disuccinate(LIII)

To a solution of astaxanthin disuccinate (XV) (0.100 g, 0.125 mmol) inDCM/DMF (3 mL/3 mL) at room temperature was added DIPEA (0.656 mL, 3.76mmol), HOBT-H₂O (0.2313 g, 1.51 mmol), DMAP (0.3067 g, 2.51 mmol), DIC(0.236 mL, 1.51 mmol), and (D)-sorbitol (0.2286 g, 1.25 mmol). Thereaction mixture was stirred at room temperature for 36 hours, at whichtime the reaction was diluted with DCM, quenched with brine/1M HCl (20mL/3 mL), and then extracted with DCM. The combined organic layers wereconcentrated to yield sorbitol diester (LIII) (44.59%) HPLC retentiontime: 8.178 min., 11.58% (AUC); LRMS (ESI) m/z (relative intensity):1148 (M⁺+Na) (40), 545 (100); HPLC retention time: 8.298 min., 33.01%(AUC); LRMS (ESI) m/z (relative intensity): 1148 (M⁺+Na) (20), 545(100), and no detectable astaxanthin disuccinate.

Example 23 Synthesis of Morpholine carbamates of astaxanthin (LIV, LV)

To a solution of astaxanthin 2E (0.100 g, 0.168 mmol) in DCM/DMF (3 mL/3mL) at room temperature was added DIPEA (0.878 mL, 5.04 mmol), DMAP(0.4105 g, 3.36 mmol), and 4-morpholine carbonyl chloride (0.196 mL,1.68 mmol). The reaction mixture was stirred at room temperature for 36hours, at which time the reaction was diluted with DCM, quenched withbrine/1M HCl (20 mL/3 mL), and then extracted with DCM. The combinedorganic layers were concentrated to yield 4-morpholine monocarbamate(LIV) (33.17%) HPLC retention time: 11.853 min., 29.01% (AUC); LRMS(ESI) m/z (relative intensity): 710 (M⁺) (100); HPLC retention time:13.142 min., 1.37% (AUC); LRMS (ESI) m/z (relative intensity): 710 (M⁺)(100); HPLC retention time: 13.383 min., 2.79% (AUC); LRMS (ESI) m/z(relative intensity): 710 (M⁺) (100), 4-morpholine dicarbamate (LV)(33.42%) HPLC retention time: 12.049 min., 29.71% (AUC); LRMS (ESI) m/z(relative intensity): 824 (M⁺+H) (54), 823 (M⁺) (100); HPLC retentiontime: 13.761 min., 1.29% (AUC); LRMS (ESI) m/z (relative intensity): 823(M⁺) (100), 692 (75); HPLC retention time: 14.045 min., 2.42% (AUC);LRMS (ESI) m/z (relative intensity): 823 (M⁺) (100), 692 (8), andastaxanthin (22.10%).

Example 24 Synthesis of Mannitol monocarbonate of astaxanthin (LVI)

To a solution of astaxanthin 2E (0.100 g, 0.168 mmol) in DCM (4 mL) at0° C. was added DIPEA (0.585 mL, 3.36 mmol), and1,2,2,2-tetrachloroethyl chloroformate (0.103 mL, 0.672 mmol). Thereaction mixture was stirred at 0° C. for 2 hours, then at roomtemperature for 1.5 hours, at which time (D)-mannitol (0.3060 g, 1.68mmol), DMF (3 mL), and DMAP (0.2052 g, 1.68 mmol) were added to thereaction. The reaction mixture was stirred at room temperature for 24hours, at which time the reaction was diluted with DCM, quenched withbrine (20 mL), and then extracted with DCM. The combined organic layerswere concentrated to yield mannitol monocarbonate (LVII) (10.19%) HPLCretention time: 9.474 min., 10.19% (AUC); LRMS (ESI) m/z (relativeintensity): 827 (M⁺+Na) (50), 804 (M⁺) (25), 725 (58), 613 (100), andastaxanthin (53.73%).

Example 25 Synthesis of (Dimethylamino)butyric acid diester ofastaxanthin (LVI)

To a suspension of 4-(dimethylamino)butyric acid hydrochloride (0.2816g, 1.68 mmol) in DCM/DMF (3 mL/3 mL) at room temperature was added DIPEA(0.878 mL, 5.04 mmol), DMAP (0.4105 g, 3.36 mmol), HOBT-H₂O (0.3094 g,2.02 mmol), DIC (0.316 mL, 2.02 mmol), and astaxanthin 2E (0.100 g,0.168 mmol). The reaction mixture was stirred at room temperature for 36hours, at which time the reaction was diluted with DCM, quenched withbrine/1M HCl (20 mL/3 mL), and then extracted with DCM. The combinedorganic layers were concentrated to yield (dimethylamino)butyric aciddiester (LVIII) (77.70%) HPLC retention time: 7.850 min., 56.86% (AUC);LRMS (ESI) m/z (relative intensity): 824 (M⁺+H) (64), 823 (M⁺) (100);HPLC retention time: 8.443 min., 3.87% (AUC); LRMS (ESI) m/z (relativeintensity): 823 (M⁺) (5), 641 (20), 520 (100); HPLC retention time:9.021 min., 16.97% (AUC); LRMS (ESI) m/z (relative intensity): 824(M⁺+H) (58), 823 (M⁺) (100), and no detectable astaxanthin.

Example 26 Synthesis of Benzyl monoether of astaxanthin (LIX)

To a solution of astaxanthin 2E (0.100 g, 0.168 mmol) and benzyl bromide(0.400 mL, 3.36 mmol) in DCM/DMF (3mL/3 mL) at 0° C. was added KHMDS(“potassium bis(trimethylsilyl)amide”) (6.72 mL; 0.5M in toluene, 3.36mmol). The reaction mixture was stirred at 0° C. for 1 hour and thenallowed to warm to room temperature. The mixture was stirred at roomtemperature for 24 hours, at which time the reaction was diluted withDCM, quenched with brine/1M HCl (20 mL/3 mL), and then extracted withDCM. The combined organic layers were concentrated to yield benzylmonoether (LIX) (15.06%) HPLC retention time: 12.705 min., 15.06% (AUC);LRMS (ESI) m/z (relative intensity): 686 (M⁺) (93), 597 (100), andastaxanthin (67.96%).

Example 27 Synthesis of Mannitol monoether of astaxanthin (LX)

To a solution of astaxanthin 2E (0.200 g, 0.335 mmol) in DCM (15 mL) atroom temperature was added 48% HBr (10 mL) and H₂O (30 mL). The aqueouslayer was extracted with DCM and the combined organic layers were driedover Na₂SO₄ and concentrated to yield the bromide derivative ofastaxanthin as a dark red oil. To a solution of the crude bromide inDCM/DMF (6 mL/6 mL) at room temperature was added DIPEA (1.58 mL, 9.09mmol), DMAP (0.3702 g, 3.03 mmol), and (D)-mannitol (0.5520 g, 3.03mmol). The reaction mixture was stirred at room temperature for 24hours, at which time the reaction was diluted with DCM, quenched withbrine/1M HCl (20 mL/3 mL), and then extracted with DCM. The combinedorganic layers were concentrated to yield mannitol monoether (LX)(4.40%) HPLC retention time: 9.479 min., 4.40% (AUC); LRMS (ESI) m/z(relative intensity): 783 (M⁺+Na) (64), 710 (66), 653 (100), andastaxanthin (79.80%).

Example 28 Synthesis of Tris(hydroxymethyl)aminomethane monoamide ofastaxanthin disuccinate (LXI)

To a solution of astaxanthin disuccinate (XV) (0.100 g, 0.125 mmol) inDCM/DMF (3 mL/3 mL) at room temperature was added DIPEA (0.653 mL, 3.75mmol), DMAP (0.3054 g, 2.50 mmol), HOBT-H₂O (0.2297 g, 1.50 mmol), andtris(hydroxymethyl)aminomethane (0.1514 g, 1.25 mmol). The reactionmixture was stirred at room temperature for 36 hours, at which time thereaction was diluted with DCM, quenched with brine/1M HCl (20 mL/3 mL),and then extracted with DCM. The combined organic layers wereconcentrated to yield tris(hydroxymethyl)aminomethane monoamide (LXI)(4.40%) HPLC retention time: 9.521 min., 3.50% (AUC); LRMS (ESI) m/z(relative intensity): 923 (M⁺+Na) (36), 900 (M⁺) (80), 560 (100); HPLCretention time: 9.693 min., 0.90% (AUC); LRMS (ESI) m/z (relativeintensity): 923 (M⁺+Na) (11), 813 (33), 500 (100), and astaxanthindisuccinate (84.34%).

Example 29 Synthesis of Tris(hydroxymethyl)aminomethane diamide ofastaxanthin disuccinate (LXII)

To a solution of astaxanthin disuccinate (XV) (0.100 g, 0.125 mmol) inDCM/DMF (3 mL/3 mL) at room temperature was added DIPEA (0.653 mL, 3.75mmol), DMAP (0.3054 g, 2.50 mmol), HOBT-H₂O (0.2297 g, 1.50 mmol), DIC(0.235 mL, 1.50 mmol), and tris(hydroxymethyl)aminomethane (0.1514 g,1.25 mmol). The reaction mixture was stirred at room temperature for 36hours, at which time the reaction was diluted with DCM, quenched withbrine/1M HCl (20 mL/3 mL), and then extracted with DCM. The combinedorganic layers were concentrated to yieldtris(hydroxymethyl)aminomethane diamide (LXII) (66.51%) HPLC retentiontime: 8.086 min., 19.34% (AUC); LRMS (ESI) m/z (relative intensity):1026 (M⁺+Na) (22), 1004 (M⁺+H) (84), 1003 (M⁺) (100), 502 (83); HPLCretention time: 8.715 min., 47.17% (AUC); LRMS (ESI) m/z (relativeintensity): 1004 (M⁺+H) (71), 1003 (M⁺) (100), 986 (62), and astaxanthindisuccinate (18.61%).

Example 30 Synthesis of Adenosine monoester of astaxanthin disuccinate(LXII)

To a solution of astaxanthin disuccinate (XV) (0.100 g, 0.125 mmol) inDCM/DMF (3 mL/3 mL) at room temperature was added DIPEA (0.653 mL, 3.75mmol), DMAP (0.3054 g, 2.50 mmol), HOBT-H₂O (0.1914 g, 1.25 mmol), and(−)-adenosine (0.3341 g, 1.25 mmol). The reaction mixture was stirred atroom temperature for 48 hours, at which time the reaction was dilutedwith DCM, quenched with brine/1M HCl (20 mL/3 mL), and then extractedwith DCM. The combined organic layers were concentrated to yieldadenosine monoester (LXIII) (21.13%) HPLC retention time: 9.005 min.,2.43% (AUC); LRMS (ESI) m/z (relative intensity): 1047 (M⁺+H) (36), 1046(M⁺) (57), 524 (100); HPLC retention time: 9.178 min., 10.92% (AUC);LRMS (ESI) m/z (relative intensity): 1047 (M⁺+H) (80), 1046 (M⁺) (100),829 (56), 524 (94); HPLC retention time: 9.930 min., 7.78% (AUC); LRMS(ESI) m/z (relative intensity): 1046 (M⁺) (100), 524 (34), andastaxanthin disuccinate (58.54%).

Example 31 Synthesis of Maltose diester of astaxanthin disuccinate(LXIV)

To a solution of astaxanthin disuccinate (XV) (0.100 g, 0.125 mmol) inDCM/DMF (3 mL/3 mL) at room temperature was added DIPEA (0.653 mL, 3.75mmol), DMAP (0.3054 g, 2.50 mmol), HOBT-H₂O (0.2297 g, 1.50 mmol), DIC(0.235 mL, 1.50 mmol), and (D)-maltose-H₂O (0.4504 g, 1.25 mmol). Thereaction mixture was stirred at room temperature for 36 hours, at whichtime the reaction was diluted with DCM, quenched with brine/1M HCl (20mL/3 mL), and then extracted with DCM. The combined organic layers wereconcentrated to yield maltose diester (LXIV) (25.22%) HPLC retentiontime: 7.411 min., 12.53% (AUC); LRMS (ESI) m/z (relative intensity):1468 (M⁺+Na) (18), 1067 (16), 827 (100); HPLC retention time: 7.506min., 12.69% (AUC); LRMS (ESI) m/z (relative intensity): 1468 (M⁺+Na)(52), 827 (76), 745 (100), and astaxanthin disuccinate (22.58%).

Example 32 Synthesis of Resveratrol esters of astaxanthin dissucinate(LXV, LXVI)

To a solution of astaxanthin disuccinate (XV) (0.100 g, 0.125 mmol) inDCM/DMF (3 mL/3 mL) at room temperature was added DIPEA (0.653 mL, 3.75mmol), DMAP (0.3054 g, 2.50 mmol), HOBT-H₂O (0.2297 g, 1.50 mmol), DIC(0.235 mL, 1.50 mmol), and resveratrol (0.2853 g, 1.25 mmol). Thereaction mixture was stirred at room temperature for 24 hours, at whichtime the reaction was diluted with DCM, quenched with brine/1M HCl (20mL/3 mL), and then extracted with DCM. The combined organic layers wereconcentrated to yield resveratrol monoester (LXV) (1.12%) HPLC retentiontime: 10.039 min., 1.12% (AUC); LRMS (ESI) m/z (relative intensity):1009 (M⁺+2H) (18), 1007 (M⁺) (21), 637 (100), resveratrol diester (LXVI)(60.72%) HPLC retention time: 10.324 min., 15.68% (AUC); LRMS (ESI) m/z(relative intensity): 1217 (M⁺) (28), 1007 (100), 609 (69), 504 (85);HPLC retention time: 10.487 min., 29.26% (AUC); LRMS (ESI) m/z (relativeintensity): 1218 (M⁺+H) (80), 1217 (M⁺) (100), 609 (60); HPLC retentiontime: 10.666 min., 15.78% (AUC); LRMS (ESI) mn/z (relative intensity):1218 (M⁺+H) (84), 1217 (M⁺) (100), 609 (71), and no detectableastaxanthin disuccinate.

Rigorous Determination of Water Solubility of the Disodium DisuccinateAstaxanthin Derivative (XVI)

A total of 30 mg of sample (disodium disuccinate astaxanthin derivative,as the all-trans mixture of stereoisomers 3S,3′S, meso, and 3R,3′R in a1:2:1 ratio) was added to 2 mL of sterile-filtered (0.2 μM Millipore®)deionized (DI) water in a 15 mL glass centrifuge tube. The tube waswrapped in aluminum foil and the mixture was shaken for 2 hours, thencentrifuged at 3500 rpm for 10 minutes. The aqueous solution wasfiltered through a 0.45 micron PVDF disposable filter device. A 1 mLvolume of filtrate was then diluted appropriately with DI water, and theconcentration of the solution was measured at 480 nM using a four pointcalibration curve prepared from fresh sample. After taking the dilutionsinto account, the concentration of the saturated solution of thedisodium disuccinate astaxanthin derivative was 8.64 mg/mL.

Experimental Data for Inhibition and/or Amelioration of DiseaseComparison of Radical-Cation Forming Ability: Non-esterified, FreeAstaxanthin and Diacid Disuccinate Astaxanthin Using Flash Photolysis

FIG. 27 and FIG. 28 depict the results of spectral analysis after flashphotolysis of the formation of triplet and carotenoid cation radicalstates for non-esterified, free astaxanthin and the diacid disuccinateastaxanthin derivative were obtained. Formation of the carotenoid cationradical is a measure of the potential biophysical behavior of the novelderivative as an antioxidant. If a derivative retains the antioxidantbehavior of non-esterified, free astaxanthin, then all previouslydocumented (i.e. literature precedent) therapeutic applications forastaxanthin can be reasonably assumed for the novel derivative,including at least singlet oxygen quenching, lipid peroxidationchain-breaking, and/or direct radical scavenging.

Irradiating carotenoids (car) directly does not result in the formationof carotenoid triplets (³car); a photosensitizer is needed. In thisexperiment, nitronaftalin (NN) was used as the photosensitizer. Afterirradiation, the excited sensitizer (NN*) forms a sensitizer triplet(³NN). When ³NN encounters a carotenoid, energy and electron transferreactions with ³NN take place. The resulting relatively stable ³car andcarotenoid cation radicals (car⁺) are detected by characteristicabsorption bands. Non-polar solvents (e.g. hexane) favor the formationof ³car, and more polar solvents (alcohols, water) favor the formationof the car⁺. The anion radical of the sensitizer (NN^(.−)) is nottypically seen because of a low absorption coefficient.

A. Spectra with Astaxanthin Disuccinic Acid (astaCOOH).

Transient Absorption Spectra of astaCOOH in Acetonitrile (MeCN),Sensitizer NN.

Negative peaks in the spectra demonstrate ground state depletion of NNand astaCOOH. The positive peak at 550 nm shows the formation of theastaCOOH triplet; the positive peak at 850 nm shows the formation of theastaCOOH cation radical. The ³car decays rather quickly. After 15 μs,half of the ³car has disappeared, and after 50 μs, no ³car is left. Thecar⁺ is stable within this time frame.

B. Spectra with Reference Compound [Non-Esterified, Free Astaxanthin(asta)].

Transient Absorption Spectra of Asta in Acetonitrile (MeCN), SensitizerNN.

The spectrum of asta is nearly identical to that of astaCOOH. After 50μs, the ³car has disappeared. During this time frame, the car⁺ isstable. Negative and positive peaks in the absorption spectra forastaCOOH and asta are superimposable.

Brief Discussion of Flash Photolysis Results:

There appears to be little difference between the diacid disuccinateastaxanthin derivative (astaCOOH) and non-esterified, free astaxandlin(asta) during flash photolysis experiments. AstaCOOH behaves like astain the flash photolysis experiments. Therefore, esterification of freeastaxanthin with succinic acid does not alter the photophysicalproperties and the cation radical lifetime. Both compounds werephotostable during the flash photolysis experiments. The disuccinateastaxanthin derivative retains the potent antioxidant potential ofastaxanthin, and is active in the esterified state. It can therefore beconsidered a “soft” drug (active as the modified entity), and not aprodrug, for therapeutic applications, conferring the valuablepropert(ies) of dual-phase radical scavenging activity to thisderivative (i.e. aqueous- and lipid-phase radical scavenging).

Induction of Connexin 43 Protein Expression

The methods for cell culture, Western blotting, quantitativedensitometric analysis, and total protein evaluation are described indetail in Rogers et al. (1990), with modifications suggested in Bertram(1999). In brief, mouse embryonic fibroblast CH3/10T^(1/2) cells weretreated with the following formulations in a 4 mL cell culture systemwith media containing 2% calf serum:

-   1. TTNPB [p-(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthyl)    propenyl benzoic acid] 10⁻⁸M in acetone [positive control for    connexin 43 upregulation (4 μl in 4 mL]-   2. Disodium salt disuccinate astaxanthin derivative/H₂O at 10⁻⁵M (40    μl in 4 mL)-   3. Disodium salt disuccinate astaxanthin derivative/H₂O at 10⁻⁶M (4    μl in 4 mL)-   4. Disodium salt disuccinate astaxanthin derivative/H₂O at 10⁻⁷M    (1:10 dilution and 4 μl in 4 mL)-   5. Disodium salt disuccinate astaxanthin derivative H₂O/ethanol    [EtOH] formulation at 10⁻⁵M (40 μl in 4 mL)-   6. Disodium salt disuccinate astaxanthin derivative H₂O/EtOH    formulation at 10⁻⁶M (4 μl in 4 mL)-   7. Sterile H₂O control (40 μl in 4 mL)-   8. Sterile H₂O/EtOH control (20 μL EtOH, 20 μL H₂O in 4 mL)-   9. Media control (4 mL)

Cells were harvested after 96 hours incubation with test compounds andcontrol solutions. All media solutions were identical in color, howeverafter treatment with the disodium salt disuccinate astaxanthinderivative at both 10⁻⁵ dilutions, the color subjectively changed to anorange-red color. Cells treated with TTNPB appeared striated with lightmicroscopy, evidence of differentiation to myocytes, an expected resultin this cell culture system. After harvest and pelleting of cells, tubescontaining both 10⁻⁵ disodium salt disuccinate astaxanthin derivativesolutions were bright red; both 10⁻⁶ dilution tubes were a pink color.As documented previously for other colored carotenoids, this wassubjective evidence for cellular uptake of the test compounds.

Cells were then lysed, and 50 μg of each protein was electrophoresed ona 10% polyacrylamide gel. The gel was then transferred to anitrocellulose filter. Total protein was assayed with Coomassie bluestaining (FIG. 29; lanes 6, 7, and 9 were smeared secondary to geltransfer, and were not included in the quantitative comparison [FIG.31)]. Western blotting was performed with anti-connexin 43 antibodiesfollowed by HRP chemiluminescence on a Biorad imager (FIG. 30). Theoriginal gel was stripped once, and the Western blot repeated twiceprior to visualization. The results were normalized to the Lane 8control (EtOH/H₂O), which demonstrates background constitutiveexpression of connexin 43 protein in a control condition (no testcompound). The results of relative connexin 43 induction by positivecontrols and test compounds are shown in FIG. 31.

Brief Discussion of Cx43 Results.

All disodium salt disuccinate astaxanthin derivative formulations testedinduced connexin 43 protein expression over the levels expressedconstitutively in water and ethanolwater controls (FIG. 31). Theprobability of detecting an induction of connexin 43 protein expressionin 5 separate test conditions in the absence of a true treatment effect(null hypothesis control μ₁=treatment mean μ₂) is 1 in 2⁵, or p=0.03.Disodium salt disuccinate astaxanthin derivatives formulated in waterinduced connexin 43 protein expression in each test condition (from 10⁻⁵to 10⁻⁷M). The decrease in the lowest disodium salt disuccinateastaxanthin derivative/water combination tested suggests dose-dependencyin the induced response. The relative induction was increased in thesingle test condition evaluated with a final ethanolic concentration inmedia of 0.5%. This finding is highly suggestive of increasedbioavailability of this formulation, as ethanol is known to reduceaggregation of disodium salt disuccinate astaxanthin derivatives inaqueous solutions. Solutions of disodium salt disuccinate astaxanthinderivatives in water at concentrations greater than 10⁻⁷ and inethanol/water combinations at 10⁻⁵ appear to have higher inductionslevels than the positive TTNPB control. TTNPB is a highly potentretinoid that is effective at inducing connexin 43 expression at the96-hour time point at 10⁻⁸M.

Induction of Intercellular Gap Junctional Communication (GJC) in MurineFibroblasts by the Disodium Salt Disuccinate Astaxanthin Derivatives

A series of experiments were performed to assess the ability of thedisodium salt disuccinate astaxanthin derivatives to induce gapjunctional communication (GJC) in an immortalized line of murinefibroblasts. Studies were conducted:

-   (1) at the functional level to measure cell/cell communication by    increased dye transfer between confluent cells in monolayer culture;-   (2) at the molecular level as measured by the ability of these    compounds to induce expression of connexin43 (Cx43) protein. Cx43 is    the structural unit of the intercellular channels in these    fibroblasts that allows GJC;-   (3) at the cellular level as shown by the ability of the disodium    salt disuccinate astaxanthin derivatives to increase the number and    size of Cx43 immunoreactive plaques in regions of the plasma    membrane in direct contact with adjacent cells.-   (1) Communication Assays. Experiments were performed to assess the    ability of the disodium salt disuccinate astaxanthin derivative [as    a statistical mixture of the all-trans (all-E) stereoisomers, S,S′,    meso, and R,R′ in 1:2:1 ratio] to enhance gap junctional    intercellular communication (GJC) between mouse embryonic fibroblast    C3H/10T1/2 cells. This ability has been previously highly correlated    with the ability of carotenoids to inhibit carcinogen-induced    neoplastic transformation (Zhang, 1992). Moreover, Cx43-mediated    junctional communication between cardiac myocytes is responsible for    transfer of signals that maintain synchronous contractions and    prevent cardiac arrhythmias (Peters, 1995).

Junctional permeability was assayed by microinjection of the fluorescentdye Lucifer Yellow CH (Sigma, St. Louis, Mo.) into individual confluentcells essentially as described previously (Zhang, 1994). Briefly,confluent cultures of C3H/10T1/2 cells were treated for 4 days with: (1)the disodium salt disuccinate astaxanthin derivative (1×10 ⁻⁵ M)dissolved in a 1:2 ethanol/water (EtOH/H₂O) formulation; (2) a syntheticretinoid, TTNPB (1×10⁻⁸M) dissolved in tetrahydrofuran as a positivecontrol; or (3) 1:2 EtOH/H₂O treated cells as a negative control. Singlecells in each dish were identified under phase contrast optics andpressure injected using a microinjection needle (Eppendorf, Hamburg,Germany) loaded with the fluorescent dye Lucifer Yellow as a 10%solution. The needle was controlled by a remote micromanipulator andcells and microscope were positioned on a pneumatic anti-vibrationtable. Successful injection of Lucifer Yellow was confirmed by briefillumination with UV light, which causes yellow fluorescence of LuciferYellow. This dye is sufficiently small to pass through gap junctions andis electrically charged, and can thus only enter cells adjacent to theinjected cell if they are in junctional communication. After 2 minutesto allow for junctional transfer, digital images were taken under UVillumination. The number of fluorescent cells adjacent to the injectedcell was later determined by digital image analysis using an unbiaseddensity threshold method and the SigmaScan software program (JandelScientific). This number of communicating cells was used as an index ofjunctional communication, as described previously (Hossain, 1993).

The results of this analysis demonstrated that the disodium saltdisuccinate astaxanthin derivative (1×10⁻⁵ M) dissolved in a 1:2EtOH/H₂O formulation effectively increased the extent of junctionalcommunication over that seen in 1:2 EtOH/H₂O treated controls. Of 22microinjected treated cells 15 (56%) were finctionally coupled by gapjunctions, in contrast to only 3 out of 11 (27%) control cells. Thesedifferences were statistically different (p<0.04; paired Student'st-test). Representative photomicrographs are shown in FIG. 14:

Panel A: treatment with the statistical mixture of stereoisomers of thedisodium salt disuccinate astaxanthin at 1×10⁻⁵M in 1:2 EtOH/H₂O;

Panel C: 1:2 EtOH/H₂O solvent negative control;

Panel E: TTNPB at 1×10⁻⁸M in tetrahydrofuran as solvent, positivecontrol; and

Panels B, D, F: digital analysis of micrographs A, C, E respectively,demonstrating pixels above a set threshold positive for Lucifer Yellowfluorescence. Because cell nuclei have the most volume, they accumulatethe most Lucifer Yellow and exhibit the most fluorescence.

-   (2) Molecular studies. Both the mixture of stereoisomers of the    disodium salt disuccinate astaxanthin derivative and purified    enantiomeric forms of the disodium salt disuccinate astaxanthin    derivative (S,S′, meso, and R,R′ forms at >90% purity by HPLC)    increase expression of Cx43 protein in murine fibroblasts as    assessed by immuno-(Western) blotting essentially as described    (Zhang, 1992 and 1994). Briefly, mouse embryonic fibroblast    C3H/10T1/2 cells were cultured in Eagle's basal medium with Earle's    salts (Atlanta Biologicals, Atlanta, Ga.), supplemented with 5%    fetal calf serum (Atlanta Biologicals, Atlanta, Ga.) and 25 μg/mL    gentamicin sulfate (Sigma, St. Louis, Mo.), and incubated at 37° C.    in 5% CO₂. On the 7^(th) day after seeding in 100 millimeter (mm)    dishes, the confluent cells were treated for four days with the    disodium salt disuccinate astaxanthin derivatives and then harvested    and analyzed for Cx43 protein induction as described. Protein    content was measured using the Protein Assay Reagent kit (Pierce    Chemical Co., Rockford, Ill.) according to manufacturer's    instructions. Cell lysates containing 100 μg of protein were    analyzed by Western blotting using the NuPage western blotting kit    and apparatus (Invitrogen, Carlsbad, Calif.) and Cx43 protein    detected using a rabbit polyclonal antibody (Zymed, San Francisco,    Calif.) raised against a synthetic polypeptide corresponding to the    C-terminal domain of mouse, human and rat Cx43. Cx43 immunoreactive    bands were visualized by chemiluminescence using an anti-rabbit    HRP-conjugated secondary antibody (Pierce Chemical Co., Rockford,    Ill.). Digital images were obtained with a cooled CCD camera, and    quantitative densitometry was then performed (Bio-Rad, Richmond,    Calif.). Equal protein loading of the lanes was confirmed by    staining with Coomassie blue protein stain and digital image    analysis.

In this experiment the disodium salt disuccinate astaxanthin derivativeswere added to cell cultures in a formulation of 1:2 ethanol/H₂O at1×10⁻⁵M. The statistical mixture of stereoisomers and purifiedenantiomeric forms demonstrated increased expression of Cx43 incomparison to cell cultures treated with 1:2 ethanol/H₂O alone (FIG. 15Aand FIG. 15B). Treatment with the statistical mixture of stereoisomersof the disodium salt disuccinate astaxanthin derivative elicited thehighest induction level of Cx43 of all derivatives tested. Theseinduction levels were several-fold less than induction levels seen withthe retinoids tetrahydrotetramethylnapthyl propenylbenzoic acid (TTNPB)(Hoffman-LaRoche, Nutley, N.J.) and retinyl acetate (Sigma, St. Louis,Mo.) included as positive controls; this relative potency difference isconsistent with previous studies.

-   (3) Cellular studies. The statistical mixture of stereoisomers of    the disodium salt disuccinate astaxanthin derivative increases    assembly of Cx43 in treated murine 10T1/2 cells in regions of    cell/cell contact consistent with formation of functional gap    junctions.

In this experiment expression and assembly of Cx43 into plaques wasassessed by immunofluorescent staining. Procedures were essentially asdescribed in Zhang (1992). Briefly, confluent cultures of C3H/10T1/2cells were grown on Permanox plastic 4-chamber slides (Nalge NuncInternational, Naperville, Ill.) and treated for 4 days with: (1) thedisodium salt disuccinate astaxanthin derivative (statistical mixture ofstereoisomers) dissolved in a 1:2 EtOH/H₂O formulation; (2) the retinoidTRNPB at 1×10⁻⁸M in tetrahydrofuran as a positive control; or (3) 1:2EtOH/H₂O as a solvent control. Cells were fixed with −20° C. methanolovernight, washed in buffer, blocked in 1% bovine serum albumin (Sigma,St, Louis, Mo.) in PBS, and incubated with the rabbit polyclonalanti-Cx43 antibody (Zymed, San Francisco, Calif.) as in (2) above anddetected with Alexa568 conjugated anti-rabbit secondary (MolecularProbes, Eugene, Oreg.). Slides were illuminated with 568 nm light andimages were acquired at a wavelength of 600 nm using the Zeiss Axioscopelight microscope and a Roper Scientific cooled CCD camera. Slidestreated with the TTNPB retinoid control and the statistical mixture ofstereoisomers of the disodium salt disuccinate astaxanthin derivative at1×10⁻⁵M exhibited assembly of immunoreactive Cx43 into plaques inregions of the cell membrane in direct contact with adjacent cells. Suchassembly is consistent with the location and formation of plaques of gapjunctions, known to be formed by the aggregation of multiple individualgap junctions in cell populations which are junctionally connected(Perkins, 1997). In cultures treated with solvent as control, suchimmunoreactive plaques were infrequent and were smaller than thosedetected in cells treated with the statistical mixture of stereoisomersof the disodium salt disuccinate astaxanthin derivative or with TTNPB aspositive control. The frequency of these plaques and their size isconsistent with the functional differences in gap junction permeabilityas detected by the Lucifer Yellow dye transfer experiments described insection 1, and FIG. 14 (TTNPB>statistical mixture of stereoisomers ofthe disodium salt disuccinate astaxanthin>solvent control), and with thedegree of induction of Cx43 as detected in the immunoblot experimentsdescribed in section 2 and FIG. 15. Representative photomicrographs areshown in FIG. 16.

Inhibition of Carcinogen-Induced Neoplastic Transformation byNon-Esterified, Free Astaxanthin in Murine Fibroblasts

Non-esterified, free astaxanthin is generated in the mammalian gut afteroral administration of esterified astaxanthin. Only free astaxanthin isfound in mammalian plasma and solid organs. This was again demonstratedin single- and multiple dose oral pharmacokinetic studies; the resultsare described herein. Inherent esterase activity of serum albumin, andthe action of promiscuous esterases in serum and solid organs rapidlygenerates non-esterified, free astaxanthin after parenteraladministration of the disodium disuccinate astaxanthin derivative (XVI).Flash photolysis experiments also demonstrated that the disodiumdisuccinate astaxanthin derivative and non-esterified, free astaxanthinhave identical antioxidant behavior in terms of formation of thecarotenoid cation radical. An experiment was performed to assess theability of non-esterified, free astaxanthin (the in vivo final cleavageproduct of the disodium salt disuccinate astaxanthin derivative (XVI),tested as the all-trans mixture of stereoisomers 3S,3S′, meso, and3R,3′R in a 1:2:1 ratio) to inhibit neoplastic transformation in the C3H10T1/2 cell culture model developed in the lab of the late CharlesHeidelberger (Reznikoff, 1973). This cell culture system has been shownto effectively mimic the initiation and transformation events of tumorformation in whole animals (Bertram, 1985). In these cells, treatmentwith the carcinogenic polycyclic hydrocarbon 3-methylcholanthrene (MCA)produces an initiation event in a small proportion of treated cells thatleads 5 weeks later to morphological transformation in these cells,exhibited by the presence of transformed foci. Injection of thesetransformed cells into syngeneic mice results in the formation ofsarcomas at the site of injection demonstrating the carcinogenic natureof the transformation (Reznikoff, 1973). This assay has been adapted tothe detection of cancer preventive agents (Bertram, 1989), and cancerpreventive retinoids and carotenoids have been demonstrated to inhibittransformation in this system (Bertram, 1991; Pung, 1988; and Merriman,1979).

This experiment was conducted according to protocols establishedpreviously (Bertram, 1991 and Pung, 1988). In brief, the 10T1/2 cells,derived from mouse embryonic fibroblasts, were seeded at a density of10³cells/60 mm dish in Eagle's Basal Media (BME) (Atlanta Biologicals,Atlanta, Ga.), supplemented with 4% fetal calf serum (AtlantaBiologicals, Atlanta, Ga.) and 25 μg/mL gentamicin sulfate (Sigma, St.Louis, Mo.). Cells were treated 24 hours later with 5.0 μg/ml MCA(Sigma, St. Louis, Mo.) in acetone or with 0.5% acetone (finalconcentration) as a control. Media was changed 24 hours after MCAtreatment. Cells were treated with astaxanthin in THF or with retinolacetate in acetone 7 days later, and re-treated every 7 days for 4weeks. Other dishes were treated with the appropriate solvent controls.After 5 weeks from the start of the experiment, cells were fixed withmethanol and stained with 10% Giemsa stain (Sigma, St. Louis, Mo.) andscored for type II and type III foci as per (Reznikoff, 1973).

The results of this analysis demonstrated that 4-week treatment withastaxanthin caused a concentration-dependent decrease in the numbers ofMCA-induced transformed foci in comparison to cells treated with MCA andwith THF as a solvent control (depicted in FIG. 34). FIG. 34 depictseffects of non-esterified, free astaxanthin (as the all-trans mixture ofstereoisomers) on MCA-induced neoplastic transformation. Graphrepresents a total of 68 cultures treated with astaxanthin at 3×10⁻⁶M,1×10⁻⁶M and 3×10⁻⁷M, delivered in a THF vehicle of 0.3%, 0.1% and 0.03%,respectively. Controls were as follows: a total of 16 dishes did notreceive carcinogen and were treated with 0.05% ethanol solvent; controlsdid not exhibit any transformation events. A total of 20 dishes weretreated with MCA and 1% THF solvent, yielding a transformation rate of0.92 foci/dish. Percent reduction (% reduction) of transformation inastaxanthin-treated dishes was calculated by a comparison of the meanfoci/dish of each treatment with the MCA-treated controls. Inferentialstatistics were performed using the paired Student's t-test; calculatedP values of 0.00004, 0.00001, and 0.00006, respectively, were obtained.P<0.05 was considered significant. Treatment with 3×10⁻⁶M astaxanthinresulted in complete suppression of the transformed phenotype (FIG. 35).FIG. 35 depicts a comparison of astaxanthin-treated dish to controldishes. Representative dishes treated with: A, no MCA with solventcontrol; B, MCA 5.0 μg/ml with 1% THF as solvent control; C, MCA with3×10⁻⁶M astaxanthin (as the all-trans mixture of stereoisomers) in THF.It is notable that this level of inhibition far exceeded that reportedpreviously for all other carotenoids tested using identical protocols(Bertram, 1991). A comparison of the current data to data previouslyreported for percent reduction in neoplastic transformation at theconcentrations tested revealed astaxanthin to be a far more potentinhibitor of transformation than either β-carotene or canthaxanthin(FIG. 36). FIG. 36 depicts a comparison of astaxanthin (as the mixtureof stereoisomers) to previously tested carotenoids. Data was compiledcomparing the percent reduction of MCA-induced neoplasticallytransformed foci/dish in cultures treated with astaxanthin to thepercent reduction of foci/dish from data previously reported by theBertram laboratory after treatment with β-carotene and canthaxanthin(Bertram, 1991) using identical protocols. The percent reduction at thehighest concentration tested previously (1×10⁻⁵M) is reported here forβ-carotene and canthaxanthin; this higher concentration of astaxanthinwas not utilized because of astaxanthin's greater measured activity atlower concentrations. These studies demonstrate the potential for thecleaved astaxanthin moiety of the synthesized derivative to be a highlyeffective cancer chemoprevention agent, after both oral and parenteraladministration. Coupled with the liver accumulation pharmacokinetic dataalso reported here (after both single- and multiple-dose strategies),the use of this compound forms a particularly useful embodiment.

Inhibition of Reactive Oxygen Species

In an experiment, neutrophils were isolated on a Percoll gradient fromwhole blood from a human volunteer. The isolated neutrophils were thenre-suspended in phosphate-buffered saline, and maximally stimulated withphorbol ester to induce the respiratory burst and production ofsuperoxide anion. To the solution of activated human neutrophils, thedisodium salt disuccinate astaxanthin derivative was added at variousconcentrations, and the superoxide signal [as measured with electronparamagnetic resonance (EPR) imaging] was subsequently measured. Thedisodium salt disuccinate astaxanthin derivative (as the mixture ofstereoisomers) reduced the measured superoxide anion signal in adose-dependent manner (FIG. 2); near complete suppression of thesuperoxide anion signal was achieved at 3 mM concentration. FIG. 2demonstrates the strong superoxide signal after activation in controls,then the results of titration with the disodium salt disuccinateastaxanthin derivative from 100 μM to 3 mM. The disodium saltdisuccinate astaxanthin derivative tested at 100 μM scavenged 28% of thetotal signal. At 3 mM, almost no superoxide signal remained. Theseresults demonstrate that cardioprotection in ischemia-reperfusioninjury, as has been demonstrated with the other anti-neutrophilinterventions described above, can also be achieved with the novelcarotenoid derivative described here. In addition to reducing thesuperoxide anion signal important in ischemia-reperfusion injury, it isalso likely that myocardial salvage can be achieved with the describednovel carotenoid derivative, as superoxide anion plays a major role intissue injury and death during prolonged myocardial ischemia.

FIG. 3 depicts an effect of a disodium salt disuccinate astaxanthinderivative/Vitamin C solution on reactive oxygen species (superoxideanion) as monitored using EPR imaging. The solution included a mixtureof about 2 to about 1 of vitamin C to disodium salt disuccinateastaxanthin derivative respectively. The disodium salt disuccinateastaxanthin derivative/Vitamin C solution reduced the measuredsuperoxide anion signal in a dose-dependent manner (FIG. 3); completesuppression of the superoxide anion signal was achieved at 0.02 μMconcentration. FIG. 3 demonstrates the strong superoxide signal afteractivation in controls, then the results of titration with the disodiumsalt disuccinate astaxanthin derivative/Vitamin C solution from 0.01 μMto 0.02 μM.

In a third experiment, neutrophils were again isolated on a Percollgradient from whole blood from a second human volunteer. The isolatedneutrophils were then re-suspended in phosphate-buffered saline, andmaximally stimulated with phorbol ester to induce the respiratory burstand production of superoxide anion. To the solution of activated humanneutrophils, the hydrochloride salt dilysinate astaxanthin derivative(XX) was added at four (4) concentrations, and the superoxide signal (asmeasured with EPR imaging) was subsequently measured. The hydrochloridesalt dilysinate astaxanthin derivative also reduced the measuredsuperoxide anion signal in a dose-dependent manner (FIG. 21), fromapproximately 5% reduction at 1 μM to 98% reduction at 3 mM. Once again,near complete suppression of the superoxide anion signal was achieved at3 mM concentration. This novel carotenoid derivative showed scavengingefficacy at low concentration (1 μM), as well as the ability forincreased concentrations of the derivative in this in vitro assay tonearly completely eliminate the superoxide anion signal. The activity ofthis novel derivative in vitro as an aqueous scavenger again suggeststhat the novel derivatives (disodium disuccinate astaxanthin,hydrochloride salt dilysine astaxanthin) will act as soft drugs (i.e.active as the intact, uncleaved novel derivatives) and not pro-drugs(inactive until cleavage to free astaxanthin) in vivo. The aqueoussolubility of this derivative (XX) was greater than 50 mg/mL,demonstrating the utility of the methods of the present invention toincrease the water solubility of the parent carotenoids (in this caseastaxanthin), from nearly zero inherent water solubility to the highmg/mL range.

Direct Superoxide Anion Scavenging by a Disodium Disuccinate AstaxanthinDerivative: Relative Efficacy of Individual Stereoisomers versus theStatistical Mixture of Stereoisomers by Electron Paramagnetic ResonanceImaging

Materials

Non-esterified, all-E astaxanthin [1:2:1 statistical mixture ofstereoisomers 3S,3′S, meso (3S,3′R and 3′S,3R), and 3R,3′R] waspurchased from Buckton Scott (India) and used as supplied (>95% purityby HPLC). Astaxanthin was dissolved in HPLC grade dimethylsulfoxide(DMSO; Sigma-Aldrich, St. Louis, Mo.). The disodium disuccinatederivatives of astaxanthin were tested separately in nine formulations:statistical mixture of stereoisomers (as for astaxanthin, above, a 1:2:1mixture of all-E; labeled as “mixture” in all tables and figures);3S,3′S, and 3R,3′R (optical isomers or enantiomers); and meso (mixtureof 3S,3′R and 3′S,3R; diastereomers of the enantiomeric pair). Alldisuccinate derivatives were synthesized at >90% purity by HPLC. Thedisuccinate derivatives were first tested at the appropriate finalconcentrations in pure aqueous solution (deionized water) from stocksolutions of 10 mM. Each of the four disuccinate derivatives were thentested from stock solutions prepared in a 1:2 mixture of ethanol (finalconcentration of EtOH in stock solution 33⅓%; final concentration inisolated neutrophil assay 0.3%; HPLC grade ethanol, Sigma-Aldrich, St.Louis, Mo.) at 10 mM. The 3S,3′S derivative was also tested from a 50%EtOH concentration stock solution (final concentration in isolatedneutrophil assay 0.5%). Ethanolic formulation of the disuccinatederivatives has been shown to completely disaggregate the supramolecularassemblies which form in pure aqueous solution, providing monomericsolutions of the derivatives immediately before introduction into thetest assay. Ethanol alone negative controls (0.3% and 0.5% final EtOHconcentrations in isolated neutrophil assay) and superoxide dismutasemimetic positive control (10 μM final concentration; Metaphore®Pharmaceuticals, Inc., St. Louis, Mo.) were also performed.

A carotenoid derivative [Succinic acidmono-(4-{18-[4-(3-carboxy-propionyloxy)-2,6,6-trimethyl-3-oxo-cyclohex-1-enyl]-3,7,12,16-tetramethyl-octadeca-1,3,5,7,9,11,13,15,17-nonaenyl}-3,5,5-trimethyl-2-oxo-cyclohex-3-enyl)ester; FIG. 17] and its stereoisomeric forms were synthesized, disodiumdisuccinate derivatives of astaxanthin, in all-trans (all-E) form. Thederivatives are symmetric chiral molecules with 2 chiral centers at the3 and 3′ carbon positions, comprising 4 stereoisomers: 3R,3′R and 3S,3′S(optical isomers, or enantiomers), as well as the diastereomeric mesoforms (3R,3′S and 3′R,3S). The statistical mixture of stereoisomerssynthesized from the commercial source of astaxanthin contains 3R,3′R,meso (3R,3′S and 3′R,3S), and 3S,3′S steroisomeric forms in a 1:2:1ratio. All individual stereoisomers and the statistical mixture weresynthesized at >90% purity by HPLC, allowing direct comparison of theindividual efficacy of these forms as direct radical scavengers. Theall-E forms of the stereoisomers used in this study were linear, rigidmolecules (bolaamphiphiles) owing to the lack of cis (or Z)configuration(s) in the polyene chain of the spacer material.

The disodium disuccinate diesters of astaxanthin demonstrate increasedwater “dispersibility” over the parent compound astaxanthin. The waterdispersibilities of the individual stereoisomers and the statisticalmixture were all greater than 8 mg/mL (approximately 10 mM), allowingthem to be introduced into the buffered aqueous test system without aco-solvent. The tendency for the parent carotenoids such as astaxanthin(Salares, 1977), as well as novel carotenoid derivatives (e.g.capsanthin derivatives) (Zsila, 2001 and Bikadi, 2002) to formsupramolecular assemblies in aqueous solution was also observed for thederivatives tested in the current study. Supramolecular self-assemblyresults in aggregates of significant size in aqueous solution, andprevents maximum direct interaction of aggregated molecules with radicalspecies. Therefore, a comparison of the direct scavenging behavior ofthe novel astaxanthin derivatives was conducted in both pure aqueousformulation as well as with the co-solvent ethanol. In stock solutions,a 1:2 concentration of EtOH/water was shown to completely disaggregatethe statistical mixture, meso, and 3R,3′R derivatives; a 50% ethanolicstock solution was required to completely disaggregate the 3S,3′Sisomer. The scavenging ability of the compounds was also tested relativeto negative (i.e. ethanol vehicle) and positive [superoxide dismutase(SOD) mimetic, free racemic astaxanthin in DMSO] controls.

Leukocyte Purification and Preparation

Human polymorphonuclear leukocytes (PMNs) were isolated from freshlysampled venous blood of a single volunteer (S.F.L.) by Percoll densitygradient centrifugation, which yielded PMNs with a purity of >95%. Each10 mL of whole blood was mixed with 0.8 mL of 0.1 M EDTA and 25 mL ofsaline. The diluted blood was layered over 9 mL of Percoll at a specificdensity of 1.080 g/mL. After centrifugation at 400× g for 20 min at 20°C., the plasma, mononuclear cell, and Percoll layers were removed.Erythrocytes were lysed by addition of 18 mL of ice-cold water for 30 s,followed by 2 mL of 10× PIPES buffer (25 mM PIPES, 110 mM NaCl, and 5 mMKCl, titrated to pH 7.4 with NaOH). Cells were pelleted at 4° C., thesupernatant was decanted, and the procedure was repeated. After thesecond hypotonic lysis, cells were washed twice with PAG buffer (PIPESbuffer containing 0.003% human serum albumin and 0.1% glucose).Afterward, PMNs were counted by light microscopy on a hemocytometer. Thefinal pellet was then suspended in PAG-CM buffer (PAG buffer with 1 mMCaCl₂ and 1 mM MgCl₂).

EPR Measurements

All EPR measurements were performed using a Bruker ER 300 EPRspectrometer operating at X-band with a TM₁₁₀ cavity. The microwavefrequency was measured with a Model 575 microwave counter (EIPMicrowave, Inc., San Jose, Calif.). To measure O₂ generation fromphorbol-ester (PMA)-stimulated PMNs, EPR spin-trapping studies wereperformed using DEPMPO (Oxis, Portland, Oreg.) at 10 mM. 1×10⁶ PMN'swere stimulated with PMA (1 ng/mL) and loaded into capillary tubes forEPR measurements. To determine the radical scavenging ability ofnon-esterified, free “racemic” astaxanthin in DMSO and the disodium saltdisuccinate derivatives in each of the nine formulations, PMN's werepre-incubated for 5 minutes with compound followed by PMA stimulation aspreviously described. The instrument settings used in the spin-trappingexperiments were as follows: modulation amplitude, 0.32 G; timeconstant, 0.16 s; scan time, 60 s; modulation frequency, 100 kHz;microwave power, 20 milliwatts; and microwave frequency, 9.76 GHz. Thesamples were placed in a quartz EPR flat cell, and spectra wererecorded. The component signals in the spectra were identified andquantified as reported (Lee, 2000).

Statistical Analysis

Statistical analyses were performed with the NCSS statistical softwarepackage (NCSS 2001 and PASS 2002, Kaysville, Utah). All statisticaltests were performed at an α=0.05.

Brief Discussion of EPR Results:

The potent SOD mimetic produced by Metaphore, Inc. served as a positivecontrol at study outset. As has been observed repeatedly in the Zweierlaboratory, the 10 μM dose in water-only vehicle nearly completelyeliminated the superoxide anion signal as detected with DEPMPO (97%inhibition; Table 1). An ethanol-alone negative control (finalconcentration 0.3%) was also evaluated, as ethanol shows minorscavenging activity in these systems; 5.7% inhibition was seen at thisconcentration. This amount of inhibition was not subtracted fromformulations containing ethanol in the descriptive data in Table 1, asthe utility of the dosing vehicle itself (disodium disuccinatederivative in EtOH) in direct scavenging was being evaluated in thisstudy. Non-esterified, free astaxanthin in DMSO (100 μM) was evaluatedas a reference standard for direct comparison to the novel derivativessynthesized for this study; mean inhibition of the astaxanthin/DMSOvehicle was 28% (Table 1).

FIG. 18 shows the relative scavenging ability of each of the 4stereoisomers (mixture and 3 individual stereoisomers) in water, at afinal concentration of 100 μM. Except for the 3R,3′R enantiomer (28.7%inhibition), all other novel derivative formulations showed decreasedscavenging ability relative to the astaxanthin/DMSO formulation (range−2.0% to 19.3% inhibition; Table 1). As can be seen, the 3S,3′Sformulation did not exhibit any mean scavenging activity. Whenintroduced into the isolated neutrophil test system in ethanolicformulation, however, in each case the scavenging ability increased overthat of the same derivative formulated in water (FIG. 19; range 38.0% to42.5%). It is important to note that the 3S,3′S derivative wasformulated in 50% EtOH for this comparison. A trend toward increasedscavenging capacity over astaxanthin in DMSO was seen for the novelderivatives in ethanolic formulation, but after subtraction of the meanscavenging ability of the ethanol vehicle (final concentration in thetest assay 0.3%), the trend was not significant (NS). In addition, nosignificant differences in mean scavenging ability were observed amongthe 4 formulations of novel derivatives tested in ethanol (FIG. 19).

FIG. 20 shows the results of titration of superoxide signal inhibitionby increasing concentrations of the mixture of stereoisomers of disodiumdisuccinate astaxanthin in ethanolic formulation. As the concentrationwas increased from 100 μM to 3 mM, near complete inhibition ofsuperoxide signal was noted (95.0% inhibition at the 3 mM dose; Table 1and FIG. 18). The dose-response curve was non-linear. Adjusting forpercent inhibition and tested dose, the disodium disuccinate derivativewas between one and two orders of magnitude less potent than the SODmimetic used as a positive control in the current study (Table 1). Table1 depicts descriptive statistics for various formulations of disodiumdisuccinate derivatives of astaxanthin tested in the current study.Sample sizes of 3 or greater were evaluated for each formulation, withthe exception of 3S, 3′S in 50% EtOH stock solution (N=2), and SODmimetic (positive control, N=1) evaluated at study outset.

Sample Solvent Concentratio N Mean (% S.D. SEM Min Max Range

TABLE 1 Mean Sample Solvent Concentration N (% inhibition) S.D. SEM MinMax Range Astaxanthin DMSO 0.1 mM 4 28.0 7.6 3.8 20 35 15 Mixture Water0.1 mM 3 19.3 0.6 0.3 19 20 1 Mixture EtOH 0.1 mM 3 38.0 8.7 5.0 32 4816 Mixture EtOH 0.5 mM 3 60.1 7.2 4.2 56 69 13 Mixture EtOH 1.0 mM 378.0 8.2 4.7 71 87 16 Mixture EtOH 3.0 mM 3 95.0 4.9 2.8 89 98 9 MesoWater 0.1 mM 3 15.7 5.9 3.4 9 20 11 Meso EtOH 0.1 mM 4 42.5 3.4 1.7 3846 8 3R, 3′R Water 0.1 mM 3 28.7 15.0 8.7 13 43 30 3R, 3′R EtOH 0.1 mM 540.8 7.5 3.3 30 50 20 3S, 3′S Water 0.1 mM 3 −2.0 4.4 2.5 −7 1 8 3S, 3′SEtOH 0.1 mM 6 21.3 4.9 2.0 15 29 14 3S, 3′S EtOH (50%) 0.1 mM 2 38 1.41.0 37 39 2 Control Water 0.0 mM 10 0.0 ND ND ND ND ND Control EtOH 0.3%final 3 5.7 2.5 1.5 3 8 5 SOD mimetic Water  10 μM 1 97.0 ND ND ND ND NDBrief Discussion of EPR Results.

Astaxanthin is a potent lipophilic antioxidant that normally exerts itsantioxidant properties in lipid-rich cellular membranes, lipoproteins,and other tissues (Britton, 1995). Derivatives of astaxanthin-withincreased utility as water-dispersible agents—have the ability todirectly scavenge aqueous-phase superoxide anion produced by isolatedhuman neutrophils after stimulation of the respiratory burst.

The pure aqueous formulations of the novel derivatives were less potentthan the ethanolic formulations in terms of direct scavenging ability.Supramolecular assembly of the water soluble carotenoid derivatives insome solvents (e.g., water) may explain their lack of potency in thosesolvents. The aggregation is of the helical, “card-pack” type, withaggregates greater than 240 nm in size forming in pure aqueous solution.Increasing ionic strength of buffer solutions may increase both the sizeand stabilility of these aggregates. The radical scavenging ability ofthese aggregates will be diminished over the monomeric solutions of thesame compounds; in fact, no scavenging ability was seen for the 3S,3′Sstereoisomer dissolved in water (Table 1, FIG. 18). Care must be takenin preparation of formulations for in vitro and in vivo testing, assupramolecular assembly limits the number of molecules available forinteraction with radical species. The size of the aggregates must alsobe taken into account, as aggregates containing as many as 10⁶ moleculesand reaching 300 nm or greater in size have been described (Bakadi,2002).

Titration of the disodium disuccinate astaxanthin derivative dose to 3mM (as the mixture of stereoisomers in 1:2 EtOH/water) demonstrated nearcomplete suppression of the superoxide anion signal (95% inhibition), asmeasured with the DEPMPO spin trap (FIG. 20). The dose-response curvewas non-linear, requiring increasing doses for near-complete suppressionof radical signal (FIG. 20). At the lowest concentration tested (100μM), nearly 40% of the signal was inhibited. The potency of the disodiumdisuccinate astaxanthin derivative at this dose can be compared directlywith the superoxide dismutase (SOD) mimetic used as a positive controlin the current study (97% inhibition at 10 μM). The results show that asan aqueous-phase radical scavenger, the disodium disuccinate astaxanthinderivative is one to two orders of magnitude less potent than the SODmimetic. However, in vivo, these derivatives decay to free astaxanthin,which becomes active in the lipid-rich membranes of cells [including themitochondrial and nuclear membranes (Goto, 2001)], therefore providingdual protection (aqueous and lipid-phase radical scavenging), notachievable with water-soluble proteins and enzyme mimetics.Non-esterified, free astaxanthin (when provided as a dietary supplementat 0.02% of feed wt/wt) is cardioprotective against the ROS-mediatedstrenuous exercise insult to both skeletal and cardiac muscle (Aoi etal. 2003). Therefore, this characteristic (i.e. dual-phase radicalscavenging) should provide additional utility for this class ofcompounds as clinical therapeutic agents in those indications for whichradical and reactive oxygen species prevention is important (Cross,1987).

The study demonstrates for the first time direct scavenging ofsuperoxide anion detected by EPR spectroscopy by a novel group ofcarotenoid derivatives. The compounds were found to form supramolecularassemblies in pure aqueous solution. Formation of supramolecularassemblies may limit their scavenging potency relative to monomericsolutions of the same compounds. No significant differences inscavenging ability were seen among the 4 potential stereoisomers of thenovel compounds. Dose-ranging studies revealed that the concentration ofderivative could be increased to near-complete suppression of theinduced superoxide anion signal. As potential in vivo therapeuticagents, this class of compounds may be used as both an aqueous phase andlipid phase scavenger, which should find wide application in those acuteand chronic disease conditions for which potent radical scavengers havedemonstrated efficacy.

Direct Superoxide Anion Scavenging by the Disodium DisuccinateDi-Vitamin C Astaxanthin Derivative

In an electron paramagnetic resonance (EPR) imaging experiment,neutrophils were isolated on a Percoll gradient from whole blood from ahuman volunteer. The isolated neutrophils were then re-suspended inphosphate-buffered saline, and maximally stimulated with phorbol esterto induce the respiratory burst and production of superoxide anion. Tothe solution of activated human neutrophils, the disodium disuccinatedi-vitamin C astaxanthin derivative (XXIII) (semi-systematic nameSuccinic acid4-[18-(4-{3-[2-(3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxy-ethoxycarbonyl]-propionyloxy}-2,6,6-trimethyl-2-oxo-cyclohex-1-enyl)-3,7,12,16-tetramethyl-octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]-3,5,5-trimethyl-2-oxo-cyclohex-3-enylester 2-(3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxy-ethylester) was added at various concentrations, and the superoxide signal(as measured with EPR imaging) was subsequently measured. The disodiumdisuccinate di-vitamin C astaxanthin derivative (XMIII) reduced themeasured superoxide anion signal in a dose-dependent manner (FIG. 33);complete suppression of the superoxide anion signal was achieved at 60μM concentration. This represents a 50-fold increase in potency over thedisodium disuccinate astaxanthin derivative (XVI) also synthesized forthe current series of experiments. The purity of the derivative astested was 88% (by HPLC area under the curve, or AUC). The novelcarotenoid derivative—designed to be a “soft-drug” by esterification tothe 6-OH position of each vitamin C—preserved the antioxidant functionof the individual vitamin C molecules. The potency of the derivative(XXIII) approached that of the formulation of disodium disuccinateastaxanthin (XVI) with free vitamin C in a 1:2 molar ratio (whichcompletely suppressed the superoxide anion signal in a 20 μM/40 μMdisodium disuccinate astaxanthin derivative (XVI)/free vitamin Cformulation). Derivative (XXIII), which generates 2 moles of freevitamin C and 1 mole of non-esterified, free astaxanthin for every moleof derivative in vivo may be particularly preferred for certain clinicalindications. Derivative (XXIII) will also likely show increased efficacyin those clinical situations in which aqueous-phase scavenging (by theintact parent derivative, as well as free vitamin C) as well aslipid-phase scavenging (by non-esterified, free astaxanthin) areimportant for reduction in the pathology attributable to ROS and otherradical species injury.

Infarct Size Reduction in Male Sprague-Dawley Rats

FIG. 4, FIG. 25, and FIG. 26 depict graphical representations of thereduction of infarct size in male Sprague-Dawley rats. MaleSprague-Dawley rats were pre-treated with the disodium salt disuccinateastaxanthin derivative (as the mixture of stereoisomers) in aqueoussolution before performing an occlusion and inducing a myocardialinfarction. Male Sprague-Dawley rats (175-200 grams) were anaesthetizedwith 100 mg/kg of Inactin, instrumented, and the heart exposed. The leftcoronary artery had a suture placed around it and was subjected to 30minutes of total coronary artery occlusion followed by 2 hours ofreperfusion, at which time infarct size was measured in hearts excisedfrom the animal. The hearts were washed in buffer and incubated intriphenyltetrazolium chloride (TTC) staining solution kept at 37° C. inphosphate buffer at pH of 7.40. Infarct size (IS) was expressed as a %of the area at risk (IS/AAR, %). Systemic blood pressure, heart rate,blood gases and body temperature were monitored throughout theexperiment, and temperature and blood gases were tightly controlled atnormal physiological levels. 25, 50, or 75 mg/kg of the disodium saltdisuccinate astaxanthin derivative or sterile saline vehicle wasadministered I.V. by tail vein injection every day for 4 days prior tothe infarct experiment and infarct size determination.

Brief Description of Salvage Results.

Infarct size reduction, and the corresponding myocardial salvage,increased linearly, and significantly, with dose (P=0.001**). At themaximum dose tested, 75 mg/kg, mean myocardial salvage was 56%, whichapproaches that achievable with ischemic pre-conditioning strategies.Volume limitations for single-dose I.V. injection in this rat precludedtesting of higher doses; however, the significant linear correlation(P<0.001**; r²=0.67) between non-esterified, free plasma levels ofastaxanthin and IS/AAR, % suggested that at doses of approximately 120to 125 mg/kg, 100% salvage might be achieved. This is, the firstdemonstration of cardioprotection by a novel carotenoid derivative.

Pharmacokinetics, Increased Bioavailability, and Increased Target TissueDistribution of the Orally Administered Disodium Disuccinate AstaxanthinDerivative

Plasma Pharmacokinetics

Single dose oral pharmacokinetic parameters (including C_(max), T_(max),AUC₍₀₋₇₂₎ V_(d), and clearance) of the disodium disuccinate astaxanthinderivative were determined in male C57BL/6 mice. The animals wereadministered the derivative orally at a single maximum dose (500 mg/kg)shown in prior studies to likely be efficacious in preventing the injurysecondary to CCl₄-administration in Sprague-Dawley rats (100 mg/kg bodyweight in those studies). Samples for HPLC analysis of levels of freeastaxanthin in plasma and liver were obtained at the following timepoints, from at least 3 animals per time point:

Time 0 [immediately before dosing of test compounds], 2, 4, 6, 8, 12,16, 24, 48, and 72 hours after ingestion.

Additional samples, with N<3, were taken at other intervals (10, 14, and36 hours; Tables 2 and 3). Non-esterified, free astaxanthin levels weredetermined in this study as carotenoid esters are completely cleaved inthe mammalian gut to free carotenoid, which moves passively across theenterocyte.

Brief Description of Experimental Methods: Plasma Pharmacokinetics

Male C57BL/6 mice, approximately 25 g, were housed in cages (threemice/cage) and fed standard mouse chow (Purina Mouse Chow, RalstonPurina, St. Louis) and water ad libitum for at least five days prior tothe start of the experiment. The disodium disuccinate astaxanthinderivative was mixed with the following components to make an emulsionsuitable for oral gavage:

-   -   Sterile filtered (0.2 micron Millipore®) water;    -   Olive oil (Bertolli USA, Inc., Secaucus, N.J.);    -   Soybean lecithin, Type IV-S (Sigma-Aldrich Co., St. Louis, Mo.        catalog number P3644).

The disodium disuccinate astaxanthin derivative demonstrateswater-solubility of approximately 8.64 mg/mL in pure aqueousformulation. In the emulsion described above, solubility was increasedto approximately 50 mg/mL, allowing for dosing up to 500 mg/kg by gavagein these animals. This significant 6-fold increase in solubility in thedosing vehicle greatly facilitated gavage studies in these small mice.

Methods for preparing the emulsion were as follows:

-   -   (1) Add 80 mg of soy lecithin (Sigma catalog P3644) to 5.0 mL        water. Vortex intermittently for approximately 30 minutes in a        15 mL centrifuge tube until the suspension is uniform;    -   (2) Add 2.5 mL olive oil at room temperature and vortex. This        produces a uniform, thick, cloudy yellow suspension. This        emulsion material may be stored either at room temperature or in        the refrigerator at 4° C. If stored, vortex immediately before        adding the disodium disuccinate derivative in Step 3 (below);    -   (3) Add the disodium disuccinate astaxanthin derivative at 50        mg/mL directly to the emulsion. The compound readily enters into        a uniform suspension at this concentration. Vortex immediately        prior to gavage to assure uniform suspension; and    -   (4) The material has the potential to clog the mouse gavage        needle. Rinse the gavage needle after every 2 gavages.

The emulsion was given by oral gavage at 500 mg/kg body weight in asingle dose. Food was withdrawn from all cages the evening prior to theexperiment. One hour after administration of the emulsion, food andwater were restored to all animals.

The methods for whole blood and tissue sampling, sample extraction, andHPLC analysis have been described in detail (Osterlie, 2000). Briefly,whole blood was collected in EDTA-containing Vacutainer® tubes, andplasma subsequently prepared by centrifigation at 4° C., 1500× g for 20minutes. Plasma samples were then aliquoted and snap frozen in liquidnitrogen prior to transport and HPLC analysis.

Tissue Accumulation

Free astaxanthin concentration was also determined, at the same timepoints as for plasma samples, in liver. Livers were removed from eachanimal in the pharmacokinetic study after sacrifice, and snap frozen inliquid nitrogen. Liver tissue was prepared for HPLC analysis asdescribed (Jewell, 1999). Therefore, simultaneous examination of liveraccumulation of free astaxanthin was performed at the same time pointsas the plasma analyses.

Brief Description of Experimental Methods: Liver Accumulation of FreeAstaxanthin

Up to 300 mg of liver from each animal was snap frozen in liquidnitrogen. Tissue homogenization and extraction were performed with amixture of chloroform/methanol/water, according to the methods of Jewell(1999). Non-esterified, free astaxanthin accumulation in liver was thenevaluated by HPLC as described above for plasma samples.

Brief Discussion of Pharmacokinetic Results

Summary tables of plasma and liver levels of free astaxanthin at theappropriate sampling interval(s) are shown as Tables 2 and 3. Plasma andliver non-esterified free astaxanthin areas under the curve vs. time(AUC's) are also included in Tables 2 and 3. The results demonstratethat for each sampling interval, the levels of free astaxanthin in liverare equal or greater to that in plasma. This improved tissue-specificdelivery to the liver is unprecedented in the literature; in fact, liverlevels of free astaxanthin are typically lower than the correspondinglevels in plasma at equivalent time points post-dose (Kurihara, 2002).Thus, the disodium disuccinate astaxanthin derivative in the emulsiondescribed above is a superior vehicle for delivery of therapeuticconcentrations of free carotenoid to tissues of interest after oraldosing.

TABLE 2 Plasma Levels of Non-Esterified, Free Astaxanthin asta asta meanTime Sample nM mg/kg mg/kg S.D. 0 PK01 0.00 0.00 PK03 0.00 0.00 PK060.00 0.00 PK15 0.00 0.00 PK16 0.00 0.00 PK20 0.00 0 2 PK10 38.04 0.02PK12 0.00 0.00 PK21 0 0 PK22 0 0 PK27 0 0 PK34 0 0 PK42 311.73 0.19 PK4374.08 0.04 PK48 48.41 0.03 PK59 318.83 0.19 0.05 0.077 4 PK07 46.18 0.03PK11 115.63 0.07 PK14 20.97 0.01 PK17 40.57 0.02 PK23 214.95 0.13 PK24179.33 0.11 PK28 PK44 80.48 0.05 PK45 67.16 0.04 PK57 119.02 0.07 PK58147.85 0.09 0.062 0.039 6 PK13 40.57 0.02 PK18 605.01 0.36 PK25 262.730.16 PK26 377.14 0.22 PK32 PK46 739.91 0.44 PK60 167.39 0.1 PK61 131.740.08 0.197 0.154 8 PK36 PK47 435.17 0.26 PK49 371.11 0.22 PK62 148.980.09 PK68 405 0.24 PK69 306.86 0.18 PK70 29.98 0.02 0.168 0.094 10 PK3112 PK37 PK63 37.19 0.02 PK64 10.93 0.01 PK67 8.12 0 PK71 53.19 0.03 PK727.66 0 PK73 8.46 0.01 0.012 0.012 14 PK51 0 0 PK52 3.14 0 0 0 16 PK658.44 0.01 PK66 10.47 0.01 PK75 28.24 0.02 PK76 4.51 0 0.010 0.008 24PK29 0 0 PK35 18.03 0.01 PK39 13.93 0.01 PK50 1.51 0 PK53 0 0 0.0040.005 36 PK38 21.37 0.01 0.01 48 PK30 0 0 PK33 0 0 PK54 22.71 0.01 PK550 0 0.003 0.005 72 PK40 1.7 0 PK41 PK56 0 0 PK74 1.92 0 0 0

TABLE 3 Liver Levels of Non-Esterified, Free Astaxanthin Time Sampleasta nM asta mg/kg mean mg/kg S.D. 0 PK01 0.00 0.00 PK03 0.00 0.00 PK060.00 0.00 PK15 0.00 0.00 PK16 7.67 0.00 PK20 8.18 0.00 0.00 0 2 PK10139.37 0.08 PK12 30.66 0.02 PK21 414.34 0.25 PK22 725.87 0.43 PK27294.07 0.18 PK34 165.32 0.1 PK42 689.36 0.41 PK43 129.66 0.08 PK48 244.50.15 PK59 564.28 0.34 0.20 0.146 4 PK07 103.07 0.06 PK11 243.4 0.15 PK1489.18 0.05 PK17 1565.15 0.93 PK19 1373.34 0.82 PK23 2558.63 1.52 PK244701.95 2.8 PK28 1023.78 0.61 PK44 359.73 0.21 PK45 211.35 0.13 PK57322.06 0.19 PK58 500.82 0.3 0.648 0.812 6 PK13 374.28 0.22 PK18 2970.441.77 PK25 3515.52 2.1 PK26 2087.8 1.24 PK32 687.99 0.41 PK46 1070.130.64 PK60 974.69 0.58 PK61 841.37 0.5 0.933 0.690 8 PK36 1290.15 0.77PK47 230.88 0.14 PK49 1115.86 0.67 PK62 1247 0.74 PK68 1263.31 0.75 PK691036.29 0.62 PK70 1518.27 0.9 0.637 0.244 10 PK31 1303.06 0.78 0.780 12PK37 3225.35 1.92 PK63 921.74 0.55 PK64 713.97 0.43 PK67 410.93 0.24PK71 1382.45 0.82 PK72 567.95 0.34 PK73 716.89 0.43 0.468 0.579 14 PK51141.9 0.08 PK52 179.51 0.09 0.085 0.007 16 PK65 240.6 0.14 PK66 340.380.2 PK75 788.66 0.47 PK76 499.84 0.3 0.278 0.144 24 PK29 440.72 0.26PK35 321.14 0.19 PK39 155.42 0.09 PK50 156.61 0.09 PK53 89.18 0.05 0.1360.086 36 PK38 658.41 0.39 0.39 48 PK30 106.07 0.06 PK33 116.79 0.07 PK5417.81 0.01 PK55 28.79 0.02 0.04 0.029 72 PK40 33.52 0.02 PK41 11.66 0.01PK56 9.21 0.01 PK74 19.31 0.01 0.013 0.005

Pre-treatment (15 days to 6 weeks) is often required when carotenoidssuch as astaxanthin are provided in oral vehicle or in feed to achieveefficacious levels in liver-injury studies (Kang, 2001; Kim, 1997; Aoiet al. 1993). In this case, therapeutic levels (200 nm or above) wereachieved with a single dose.

The Cmax (Table 4) of 0.9 mg/L is also unprecedented in rodents, animalswhich absorb only a small percentage of the oral dose of carotenoids. Itis significant that these plasma and liver levels of free carotenoidwere obtained after just a single dose of compound in the emulsionvehicle. In humans, Osterlie et al. (2000) have described Cmax plasmalevels of 1.3 mg/L after a single dose of 100 mg (approximately 1.1mg/kg oral dose) of non-esterified, free astaxanthin in olive oilvehicle. Humans typically absorb 40 to 50% of the oral dose ofcarotenoid when provided in fatty vehicle, as opposed to a fewpercentage points for rodents. Therefore, the current study demonstratesachievement of nearly 70% of the Cmax in humans with the emulsionvehicle developed for rodents, greatly increasing the utility of thisderivative for hepato-protection studies.

TABLE 4 pK Parameters Parameter Liver Plasma *Cmax (mg/L) 0.9 0.2 **Tmax(hr) 6 6 Elimination half-life (hr) 11.655 3.938 Elimination rate (l/hr)0.059 0.176 ***AUC(0-72) (mg hr/L) 15.8 1.2 ***AUC□ (mg hr/L) 15.9 1.2Oral clearance (L/hr) 15.856 216.822 Volume of distribution (L/kg) 263.91232.1 *Maximal concentration **Time at maximum concentration ***Areaunder the curve

Reduction of Experimental Infarct Size and Circulating Levels ofC-Reactive Protein in Rabbits After Parenteral Administration of Cardax™(Disodium Disuccinate Astaxanthin Derivative)

The influence of parenteral administration of the disodium disuccinateastaxanthin derivative (XVI) on induced infarct size and induced levelsof circulating C-reactive protein (CRP) in rabbits was investigatedusing the methods of Barrett et al. (2002) with slight modifications.The purpose of the current study was to investigate the ability of thedisodium disuccinate astaxanthin derivative (XVI) to reduce inflammationas measured by CRP in the setting of experimental myocardialischemia/reperfusion injury in the rabbit heart. It has been suggestedthat CRP, commonly used as a marker for the acute inflammatory(“acute-phase”) response, may actually have a pro-inflammatory effectmediated through the activation of the complement cascade. Myocardialischemia/reperfusion injury, which is accompanied by an increase in theformation of oxygen radicals (ROS), has also been shown to activate thecomplement system. It has been demonstrated that (1) the endogenousincrease in plasma CRP secondary to a remote inflammatory lesion wasassociated with an increase in myocardial tissue injury secondary toregional ischemia and reperfusion; (2) this increase in injury(manifested as increased infarct size) was mediated by complementactivity; and (3) CRP was an “effector”, and not merely an indirectmeasure of systemic inflammation, in this system. Therefore, reductionof circulating CRP levels, together with the reduction(s) in infarctsize previously noted with Cardax™ in rodents, would form a powerfulanti-inflammatory therapeutic modality in the acute coronary syndromesetting.

In brief, male New Zealand white rabbits (2.25-2.5 kg) were used for thestudy. The acute phase inflammatory response was induced by subcutaneousinjection of four aliquots (0.5 mL each) of 1% croton oil in corn oilbeginning on the second day of pre-treatment with Cardax™. EitherCardax™ (at 50 mg/kg IV by ear vein injection) in water or equal volumesof sterile saline were given once per day for 4 days prior toexperimental infarction on day 5. The time course of increases incirculating CRP levels were obtained as described previously (Barret etal. 2002), using an ELISA-based method with anti-rabbit CRP antibodies.On the final day of the experiment (day 5: approximately 24 hours afterthe last drug infusion), the rabbits were anesthetized with a mixture ofxylazine (3 mg/kg) and ketamine (35 mg/kg) followed by pentobarbital (90mg/kg) intramuscularly. Additional pentobarbital was administered asnecessary to maintain anesthesia. After tracheotomy, the rabbits wereventilated with room air, and the heart was exposed via a leftthoracotomy. The heart was then supported in a pericardial cradle and a3-0 silk ligature was placed around the left anterior descendingcoronary artery. The artery was occluded for 30 minutes by exertingtraction on the ligature and subsequently reperfused for 180 minutes.Shortly before completing the protocol, a venous blood sample wasobtained for determination of plasma CRP.

At the completion of the reperfusion phase of the protocol, the heartswere removed and cannulated by the aorta on the Langendorff perfusionapparatus. The hearts were then perfused with a modified Krebs-Henseleitbuffer for 10 to 15 minutes (20-25 mL/minute). At the conclusion of thisperiod, the hearts were perfused with 80 mL of 0.4%2,3,5-triphenyltetrazolium chloride (TTC) at 37° C. for determination ofthe area-at-risk (AAR). The left circumflex coronary artery was thenligated in the same area as it was during the surgicalpreparation/experimental infarction. At this time, the perfusion pumpwas stopped, and 3.0 mL of Evan's blue dye was injected slowly into thehearts through a sidearm port connected to the aortic cannula. Thesolution was allowed to distribute through the heart for approximately30 seconds. The hearts were then cut into six transverse sections atright angles to the vertical axis. The right ventricle, apex, and atrialtissue were discarded. Tissue demarcated by a purple/blue colorrepresented the region perfused by the noninfarct-related coronaryartery distribution. Both surfaces of each transverse section weretraced onto clear acetate sheets that were scanned and subsequentlydigitized to calculate infarct area. Total area at risk was expressed asa percentage of the left ventricle. Infarct size was then expressed as apercentage of area at risk.

Mean infarct size in control animals and Cardax™—treated animals isshown in FIG. 37. Levels of circulating CRP in control animals andCardax™—treated animals (shown as the mean difference between baselinelevels and induced levels at the time of reperfusion) is shown in FIG.38. Reductions in infarct size of approximately 55.4% percent were seenin Cardax™—treated rabbits; ischemic area at risk was similar in bothgroups. Similarly, the mean increase in circulating CRP levels incontrols (+23.5%) over baseline was completely abrogated in theCardax™—treated animals, to mean levels below those observed at baseline(−15.7%). As CRP is both an effector in the acute coronarysyndrome—resulting in an increased infarct size in the presence ofelevated levels of this acute phase reactant—and a strong independentpredictor of cardiovascular risk in primary and secondary preventioncardiac patients—reductions in the levels of this circulating proteinforms a strong therapeutic modality.

Oral Administration of Disodium Disuccinate Astaxanthin Reduces AlanineAminotransferase (ALT) Elevations Produced by Lipopolysaccharide (LPS)in Mice

The following study evaluates the utility of oral administration of thedisodium disuccinate astaxanthin derivative for hepatoprotective effectsin a model of LPS-induced liver injury in mice.

Brief Description of Experimental Methods:

Three-month old male ICR mice were treated with LPS and galactosamine inorder to induce liver injury (Leist, 1995). Mice were first orallygavaged with either an olive oil/water/lecithin emulsion (10 mL/kg, or0.3 mL for a 30 gram mouse), or the same emulsion containing thedisodium disuccinate astaxanthin derivative (50 mg/mL) for a finaldisodium disuccinate astaxanthin dose of 500 mg/kg. Two hours later micewere injected intraperitoneally (IP) with either saline (10 mL/kg) or asolution of E. coli LPS (3 mg/kg, Sigma catalog number L-3755) andD-galactosam (700 mg/kg). Animals were sacrificed by carbon dioxide(CO₂) asphyxiation 5 hours after the IP injection, and plasma was thencollected for ALT determination.

Brief Description of LPS-Induced Injury Results.

These initial results demonstrated that the disodium disuccinateastaxanthin derivative had no effect on plasma ALT in the salineinjected (liver-injury sham-treated control) animals. In control animalsgavaged with emulsion only (without the derivative), there was a greaterthan 3-fold increase in ALT. In animals that received the emulsion withdisodium disuccinate astaxanthin derivative at 500 mg/kg included, theALT elevation was substantially reduced (N=3 animals per group),demonstrating the efficacy of the compound in reducing ALT, a serummarker of hepatocyte necrosis in these animals. As LPS-induced liverinjury is mediated by ROS (including the radical nitric oxide NO.), andsubstantial systemic inflammation occurs after LPS insult, for whichnon-esterified, free astaxanthin is protective (Ohgami et al. 2003), theutility of the novel derivative for clinical indications in which suchinflammation is promoted represents a particularly useful embodiment.

Accumulation of Free Astaxanthin in Plasma and Liver After Multiple DoseOral Administration in Black Mice

In this pharmacokinetic study, with methods as described herein, eleven(11) individual daily oral doses of the disodium disuccinate astaxanthinderivative (500 mg/kg) were given by oral gavage in the emulsion vehicleto black mice, and the accumulation of free astaxanthin in plasma andliver was measured in three (3) animals at the probable Cmax and Tmax (6hours). Probable Cmax and Tmax (6 hours) was deduced from plasma andliver samples in the prior single dose oral pharmacokinetic study.Accumulation of non-esterified, free astaxanthin in plasma and liverafter single emulsion doses was assessed. The mean plasma concentrationfor all animals tested was 381 nM. Mean liver concentration for allanimals tested was 1735 nM. In the single dose study, on average, aprotective level (set at the antioxidant ED₅₀ for non-esterified, freeastaxanthin of 200 nM) was achieved in both plasma and liver; the meanliver concentration achieved was almost 9 times the protective level.

In the multiple dose study, both peak and trough levels were taken (peaklevels taken 6 hours after dosing at the probable Cmax; trough levelsobtained 6 hours after Cmax, or 12 hours post-dose). Mean peak levels inplasma at peak and trough, respectively, were 485 nM and 231 nM; meanpeak levels liver at peak and trough, respectively, were 1760 nM and 519nM. Again, in each case protective levels were achieved and maintainedto 11 days post-multiple dosing; in the case of liver, levels almost 9times the protective level were achieved. Again, at each time pointafter multiple dosing, the accumulation in liver was greater than thatobserved in plasma, demonstrating the increased utility of this dosingvehicle for targeting to this solid organ (FIG. 32). It is also apparentfrom this data set that chronic administration of the disodiumdisuccinate astaxanthin derivative will be efficacious inhepatoprotection.

Accumulation of Free Astaxanthin in Myocardium (Heart) and Brain AfterSingle Dose Oral Administration in Black Mice

A single maximum dose of the disodium disuccinate astaxanthin derivative(500 mg/kg) was given by oral gavage in the emulsion vehicle to blackmice, and the accumulation of non-esterified, free astaxanthin wasmeasured in four (4) animals at the probable Cmax and Tmax (6 hours), asdeduced from plasma and liver samples in the prior study. Accumulationof non-esterified, free astaxanthin in heart after a single dose wasexcellent (mean+/−SEM of 4 animals=693.25+/−272 nM), and paralleled thatseen with accumulation of non-esterified, free astaxanthin in liver.Again, at each time point, the accumulation in heart was greater thanthat observed in plasma, demonstrating the increased utility of thisdosing vehicle for targeting to solid organs. Accumulation ofnon-esterified, free astaxanthin in the CNS (brain) was less striking(mean+/−SEM of 4 animals=3.6+/−1.7 nM), suggesting that penetration ofthe blood-brain barrier (BBB) was possible, but that chronic,multiple-dose administration may be necessary to achieve protectivelevels for those CNS applications (Alzheimer's disease, stroke, etc.).

Interaction of the Disodium Salt Disuccinate Derivative ofMeso-Astaxanthin with Human Serum Albumin (HSA)

Poor aqueous solubility of most carotene carotenoids, and the vastmajority of xanthophylls limits their use as aqueous-phase singletoxygen quenchers and radical scavengers. Chemical modifications whichincrease the apparent solubility and/or dispersibility of thecarotenoids have found application in basic science as well as clinicalresearch. However, the tendency for the parent carotenoids and novelderivatives to form supramolecular assemblies in aqueous solutionwarrants comprehensive evaluation of such behavior prior to moving intoin vitro and in vivo assays of the efficacy of such compounds.

FIG. 5 depicts a carotenoid derivative, the disodium salt disuccinatederivative (DAST) of synthetic meso-astaxanthin(3R,3′S-dihydroxy-β,β-carotene-4,4′-dione), in all-trans (all-E) form.The symmetric C₄₀-xanthophyll used to generate the new derivative hastwo chiral centers at the 3 and 3′ positions. In aqueous solutionC₄₀-xanthophyll exhibits no optical activity, as these stereocentershave opposite absolute configurations and internally compensate eachother. Natural carotenoid molecules possessing carboxylic functionalitybind preferentially to human serum albumin (HSA), the most abundantprotein in the blood. Since albumin binding strongly influences thepotential in vivo biochemical activities of a given compound, circulardichroism (CD), ultraviolet-visible (UV/Vis) and fluorescencespectroscopy were used to characterize the interaction of this novelcarotenoid derivative with fatty acid-free HSA. The protein binding andaggregation properties were investigated of this symmetric carotenoidattached through direct esterification to a moiety with carboxylate endgroups, forming a rigid, long-chain, highly unsaturated dianionicbolamphiphile. It was verified that in buffer solution in the absence ofprotein, the meso-carotenoid formed closely-packed H-type (card-pack)aggregates exhibiting no CD Cotton effects (CE). At low ligand/protein(L/P) molar ratios, however, the meso-carotenoid immediately andpreferentially associated with HSA in monomeric fashion, suggesting thatthe secondary chemical interactions (van der Waals forces, hydrogenbonding) that permit supramolecular assembly in aqueous solution wereovercome in a biologically relevant environment. Above 1:1ligand/protein molar ratio the meso-carotenoid molecules again began toaggregate; the aggregation observed at these ratios was chiral,resulting in a supramolecular structure showing intense, exciton-type CDactivity.

Brief Description of Experimental Methods

The novel derivative dAST was synthesized from crystalline astaxanthin[3R,3′R, 3R,3′S, 3S,3′S (25:50:25)], a statistical mixture ofstereioisomers obtained commercially (Buckton Scott, India). Theastaxanthin stereoisomers were separated by high-pressure liquidchromatography (HPLC), allowing for the synthesis of the purifiedmeso-disodium salt disuccinate derivative for testing in the currentstudy. The all-trans (all-E) form of the meso stereoisomer used was alinear, rigid molecule owing to the lack of cis (or Z) configuration(s)in the polyene chain of the spacer material (FIG. 5). The disodium saltdisuccinate derivative of synthetic meso-astaxanthin was successfullysynthesized at >99% purity by HPLC.

Materials

Essentially fatty acid-free human serum albumin (catalog No. A-1887, lotNo. 14H9319) were obtained from Sigma and used as supplied.Double-distilled water and spectroscopy grade dimethyl sulfoxide (DMSO,Scharlau Chemie S. A., Barcelona, Spain) and ethanol (Chemolab,Budapest, Hungary) were used. All other chemicals were of analyticalgrade.

Preparation of Stock Solution of dAST

After dissolution of the meso-carotenoid in DMSO, 100 μl of DMSOsolution was added to 2 mL ethanol in a rectangular cuvette with 1 cmpathlength. The absorption spectrum was registered between 260 and 650nm. Concentration was calculated from the light absorption value at theλ_(max) (ε_(478 nm)=116,570 M⁻¹ cm⁻¹).

Preparation of HSA Solutions

For spectroscopic sample preparation, HSA was dissolved in pH 7.4 Ringeror 0.1 M pH 7.4 phosphate buffer solutions. Albumin concentration wascalculated with the value of

E_(1cm)^(1%) = 5.31,using experimentally obtained absorbance data at 279 nm. The molecularweight of HSA was defined as 66500 Da.Circular Dichroism and UV/Vis Absorption Spectroscopy

CD and UV spectra were recorded on a Jasco J-715 spectropolarimeter at25±0.2 and 37±0.2° C. in a rectangular cuvette with 1 cm pathlength.Temperature control was provided by a Peltier thermostat equipped withmagnetic stirring. All spectra were accumulated three times with abandwidth of 1.0 nm and a resolution of 0.5 nm at a scan speed of 100nm/min. Induced CD was defined as the CD of the dAST-HSA mixture minusthe CD of HSA alone at the same wavelengths, and is expressed asellipticity in millidegrees (mdeg).

CD/UV/Vis Titration of HSA with dAST in pH 7.4 Ringer and 0.1 MPhosphate Buffer Solutions at 37° C.

Ringer buffer, L/P values from 0.007 to 0.10: 2 mL of 1.6×10⁻⁴M HSAsolution was placed in the cuvette with 1 cm optical pathlength andsmall amounts of the ligand stock solution (c=2.2×10⁻⁴) were added withan automatic pipette in 10 μL aliquots. Ringer buffer, L/P values from0.82 to 13.13: 2 ml of 2.3×10⁻⁶M HSA solution was placed in the cuvettewith 1 cm optical pathlength and μL volumes of the ligand stock solution(c=3.9×10⁻⁴) were added with an automatic pipette. Phosphate buffer, L/Pvalues from 0.82 to 13.10: 2 mL of 2.2×10⁻⁶M HSA solution was placed inthe cuvette with 1 cm optical pathlength and μL volumes of the ligandstock solution (c=3.6×10⁻⁴) were added with an automatic pipette.

Measurement of the Intrinsic Fluorescence of HSA in the Presence of dAST

2 mL of 4.2×10⁻⁶M HSA solution was prepared in a 1 cm rectangular cellin 0.1 M pH 7.4 phosphate buffer. 1.3×10⁻⁴ and 3.3×10⁻⁴M meso-carotenoidDMSO solutions were consecutively added in μL volumes to the cuvette inthe sample chamber of the Jasco J-715 spectropolarimeter. The resultingsample solution was excited between 240 and 360 nm in 0.5 nm wavelengthincrements. Total fluorescence intensity was collected at eachwavelength with a Hamamatsu H5784-type photomultiplier detector mountedon a right angle to the light source. In the sample solution, initialand final concentrations of HSA and dAST were 4.2×10⁻⁶M−4.0×10⁻⁶M and1.3×10⁻⁷M−1.4×10⁻⁵M, respectively. The meso-carotenoid/HSA molar ratiowas varied between 0.03 and 3.53. During the fluorescence measurements,final DMSO concentration did not exceed 5 v/v%. A control experiment wasalso performed, in which the fluorescence of HSA during addition of 20,50 and 100 μL DMSO to the solution was measured.

Brief Discussion of UV/Vis and CD Spectroscopy Results

UV/Vis and CD Spectral Properties of dAST in Ethanol and Aqueous BufferSolution

Because of its extended π-system, dAST exhibited intense lightabsorption in the visible spectrum (FIG. 6). The main bell-shapedabsorption band centered at 481.5 nm was due to the lowest energyelectronic dipole allowed, a π→π* transition polarized along the longaxis of the polyene chain. At room temperature, lack of fine structureis typical for carotenoids containing one or more conjugated carbonylgroups. However, the vibrational sub-bands were indeed present beneaththis curve, as revealed by the second derivative of the spectrum (FIG.6). Additionally, in the near-UV region, further transitions werepresent. According to theoretical calculations performed on polyenemodels, the electronic transition moment (μ) of the moderately intenseband around 300 nm is polarized parallel to the long axis of the dASTmolecule. At the same time, the band at 371 nm μ is oriented along thetwofold, C₂ symmetry axis of the conjugated system. The weak n→π*transitions of the carbonyl groups were obscured by the other bands. Asexpected, the meso-carotenoid compound did not show any CD bands inethanol since the effects of the two opposite chiral centers (3R,3′S)canceled each other (data not shown).

In Ringer buffer solution, the principal absorption band of DASTchanged, exhibiting a large blue-shift (2541.6 cm⁻¹) as well asbandwidth narrowing (FIG. 7). These spectral changes indicated theformation of so-called “card-pack” aggregates, in which the moleculeswere held together in close proximity (within a few angstroms) by bothexclusion from the aqueous environment and H-bonding interactions. As aresult, the excited-state wave functions of the polyene chains weredelocalized inter-molecularly, allowing exciton resonance interaction tooccur between neighboring molecules. This interaction resulted in ahigh-energy exciton peak in the UV/Vis spectrum. Due to unfavorablesteric interactions arising among the bulky end-groups, parallelalignment of the polyene chains is not allowed; the long axes of theseparate molecules instead close a definite intermolecular overlayangle. In such cases, carotenoid aggregates built up by chiral monomersalso exhibit induced Cotton effects (CE) due to the chiralintermolecular arrangement determined by asymmetric centers. Incontrast, the meso-carotenoid compound demonstrated no optical activityin the aggregated state in solution (data not shown) due to the lack ofnet chirality of the molecules.

Optical Properties of dAST in the Presence of Human Serum Albumin at LowLigand/Protein Molar Ratios

Upon addition of dAST to the HSA solution prepared in pH 7.4 Ringerbuffer, two definite, oppositely-signed induced CD bands appearedbetween 300 and 450 nm with a zero cross-over point at 367 nm (FIG. 8).The figure inserts show the intensities of the induced Cotton effectsand the main absorption band at different L/P ratios (Δε and ε valuesare calculated with respect to the total meso-carotenoid concentration).Magnitudes of the CEs increased with increasing concentration of theligand, however, their shape and wavelength positions remain unchanged.As mentioned above, there are two transitions below 450 nm which mightbe responsible for the observed optical activity. The absorption bandaround 300 nm has transition symmetry B, and the corresponding electricand magnetic transition moments are perpendicular to the twofoldsymmetry axis along the polyene chain. The electric and magnetictransition moments of the band at 372.5 nm are polarized parallel to theC₂ axis, its transition symmetry is A. It is reasonable to assume thatupon protein binding, these bands shift to longer wavelengths due to thechanging microenvironment surrounding the polyene chain. It has beenwell established that CD spectra of carotenoids in which thechromophoric portions belong to the C₂ point group conform to theC₂-rule: if the overall conjugated system acquires right-handedchirality (i.e. dihedral angles around bonds 6-7 and 6′-7′are negative),then transitions of symmetry A lead to negative CE, and transitions ofsymmetry B lead to positive CE (FIG. 8). Therefore, the meso-carotenoidbinds to HSA in such a manner that the protein environment fixes theterminal rings in a well-defined chiral conformation that results in theobserved negative- and positive-induced CD bands.

The absolute configurations of the chiral 3 and 3′ centers do notdetermine the chiroptical property of the molecule; rather, theasymmetric protein environment of the albumin molecule (via non-covalentchemical interactions) determines the observed activity. In contrast tothe aggregate behavior in the aqueous solutions described above, thedAST molecules do not aggregate in HSA solution at these L/P ratios, asdemonstrated by the retention of the bell-shaped and slightlyred-shifted visible absorption band (FIG. 8). Thus, both the UV/Visabsorption and CD spectra indicate that the binding of themeso-carotenoid molecules to HSA occurs in monomeric form.

Optical Properties of dAST in the Presence of HSA Above 1:1 L/P Ratios

An increasing amount of dAST was added to solutions of HSA preparedeither with pH 7.4 Ringer or 0.1 M pH 7.4 phosphate buffer to achieveL/P ratios higher than 1. Both CD and UV/Vis absorption spectraexhibited profound changes during addition of the ligand (FIG. 9 andFIG. 10). In addition to the blue-shifted visible absorption band a new,positive-negative CD band pair appeared around 480 and 420 nm,respectively. These CE's exhibited no vibrational fine structure andtheir amplitudes grew with increasing concentration of the ligand.However, there were some notable differences between the spectraobtained in the Ringer and phosphate buffer solutions:

-   -   a) The main absorption band shifted to lower wavelength (434.5        nm) in Ringer buffer. The corresponding value was 451.5 nm in        phosphate buffer.    -   b) Deviation of the zero cross-over point of CEs from the        maximum of the absorption band was three times larger in Ringer        (441.6 cm⁻¹) than phosphate buffer solution (148.4 cm⁻¹).    -   c) Above an L/P value of 8, the intensities of the CD bands no        longer increased in Ringer solution. In contrast, the        amplitude(s) of the CD bands continued to increase with        increasing L/P ratio in phosphate buffer, even at an L/P value        of 13.    -   d) At the same L/P ratios, more intense CD bands were measured        in phosphate buffer (FIG. 9 and FIG. 10).        The fact that these oppositely-signed CD bands appear only above        1:1 L/P ratio strongly suggests that they stemmed from chiral        intermolecular interactions between adjacent meso-carotenoid        molecules. When two electric transition dipole moments are        similar in energy, lie close to each other in space, and form a        chiral array, their interaction is manifested as chiral exciton        coupling: the CD spectrum shows a bisignate couplet matched with        the spectral position of the corresponding absorption band,        whose sign is determined by the absolute sense of twist between        the two dipoles. According to the exciton chirality rule, a        positive twist corresponds to a positive long-wavelength CE and        a negative CE at shorter wavelength, and vice versa. In our        case, the direction of the transition dipole moment is known; it        is polarized along the long axis of the polyene chain. Thus, the        neighboring meso-carotenoid molecules are arranged in such a        manner that their long axes form a positive (clockwise)        intermolecular overlay angle. Chiral arrangements of two        conjugated chains shown in FIG. 11 satisfy the former condition;        in these cases, a long-wavelength positive and a short        wavelength negative band would appear in the CD spectrum.        However, the spectroscopic behavior of the absorption band helps        to differentiate between these spatial arrangements. Due to        unfavourable Coulombic interactions between the transition        dipole moments of neighbouring meso-carotenoid molecules in the        case of a and b (FIG. 11), the absorption maximum shifts to        higher energies; if the c form exists, then the absorption band        widens and its maximum shifts to lower energies. Consequently,        DAST molecules form a right-handed chiral array in which the        long axes of meso-carotenoid monomers form an acute, positive        angle (FIG. 11, a and b).

The following scenario is proposed for the origin of the chiral orderingof the ligand molecules. Albumin appears necessary for the inducedoptical activity and, at first, it is tempting to assume that there is alarge binding site on HSA able to accommodate two meso-carotenoidmolecules. At low L/P values albumin would bind only a single ligand; athigher L/P concentrations, a second meso-carotenoid monomer would becomplexed. As stated above, however, the magnitudes of CEs continue toincrease at quite high L/P values (FIG. 10), in which case a singlebinding site should already be saturated. One resolution to this issueassumes that HSA is an asymmetric template on which the chiralself-assembly is started. The first few meso-carotenoid molecules bindto HSA in right-handed arrangement, and subsequent meso-carotenoidmonomers build upon this chiral architecture. In this scenario, HSAprovides the first essential step, the chiral initiation (“chiralseeding”); after this the self-assembly continues automatically. It isvery important to note, however, that without their chiral end-groupsonly a few dAST molecules would be held in right-handed arrangement atthe binding site of HSA. The 3 and 3′ chiral centers play a decisiverole in allowing the aggregates to form the chiral self-assembly on theHSA molecules. In the absence of protein, the meso-carotenoid moleculesform right- and left-handed assemblies to an equal extent, due to thelack of chiral discrimination.

As listed above, the spectral differences between the CD curves measuredin phosphate buffer and Ringer solutions suggested the influence of thesalt concentration on the stability of the aggregates (FIG. 9 and FIG.10). The osmolarity and ionic strength of the Ringer buffer was higherthan that of phosphate buffer. The succinic moieties were ionized at pH7.4 in both buffer solutions and electrostatic repulsion arose bothwithin and among the aggregates. Positively-charged salt ions are ableto decrease this repulsion, and therefore contribute to an increasingstability and size of the aggregates in the presence of these cations.During the titration of HSA with DAST above the 1:1 L/P ratio, bothchiral and achiral aggregates were simultaneously formed; however, onlychiral aggregates were associated with HSA, while achiral aggregateswere not. CD spectra obtained in Ringer buffer solution (FIG. 9)suggested that the achiral aggregates were better stabilized in thishigher osmolarity buffer due to the screening effect of the salt ions.The added ligand molecules preferentially associated with existingaggregates, which resulted in the amplitudes of the CD bands reaching aplateau and becoming constant in contrast with the phosphate buffer.

Fluorescence Quenching of HSA Upon Addition of dAST

The single tryptophan residue (Trp214) located in the depth of subdomainIIA is largely responsible for the intrinsic fluorescence of HSA. Thefluorescence emission spectrum of HSA overlaps with the absorptionspectrum of the meso-carotenoid. Therefore, fluorescence spectroscopicmeasurements were obtained after incremental addition of DAST in DMSO toa solution of HSA. The results clearly demonstrated that themeso-carotenoid molecules were able to effectively quench the intrinsicfluorescence of HSA (FIG. 12). The DMSO used to prepare the stocksolution of DAST exhibited a negligible effect on the intrinsic HSAfluorescence (FIG. 12). At an L/P ratio of 0.7, the baselinefluorescence intensity decreased by 50%. The observed phenomenonsuggested that a meso-carotenoid molecule was bound in the vicinity ofTrp214, which forms part of the wall in one of the two main bindingcavities of HSA (site I, subdomain IIA; FIG. 13). However, neither siteI nor site II (subdomain IIIA)—both hydrophobic fatty acid bindingtunnels—are capable of accommodating the long, rigid dAST molecule (FIG.13). Based on structural similarity, a second possibility is that dASTbinds to other long-chain (C18, C20) fatty acid binding sites of HSA,which have been well-characterized by high resolution X-raycrystallography. In the case of shorter, open-chain carotenoids havingno bulky end-groups, this possibility may be likely. However, thepolyene chain of the meso-carotenoid derivative itself measures 28 Å(between the 3 and 3′ chiral carbon atoms). Despite their conformationalmobility, the succinate moieties require additional space, increasingthe effective length of the molecule to 48 Å. Careful inspection of thecrystal structure of HSA suggests that the long, narrow cleft betweendomains I and III may be suitable for the binding of a meso-carotenoidmolecule (FIG. 13). The interdomain cleft is wide, and its narrow end isclose to the tryptophan (Trp214; * on FIG. 13) residue which wouldprovide a structural explanation for the observed fluorescence quenchingupon binding of the meso-carotenoid molecule to the interdomain cleft ofHSA. Furthermore, it may be assumed that association of additional dASTmolecules to the single one in the interdomain cleft induces significantconformational changes of HSA resulting in the widening of the centralcrevice. This might be the reason why the fluorescence quenching did notstop at an L/P=1 ratio but keeps on strengthening as the CEs increase(FIG. 13).

Discussion of UV/Vis and CD Spectroscopy Results

As a consequence of exclusion from the aqueous environment andintermolecular hydrogen bonding, the disodium salt disuccinatederivative of synthetic, achiral meso-astaxanthin formed opticallyinactive, card-pack type aggregates in aqueous buffer solutions, asindicated by the large blue-shift of the main visible absorption bandversus the band observed in ethanolic solution. In the presence of anexcess of fatty acid-free HSA, the meso-carotenoid appears to bepreferentially associated with HSA in monomeric fashion. These resultssuggest that the weak van der Waal's forces and hydrogen bonding thatpermits supramolecular assembly in aqueous solution will be rapidlyovercome in a biologically relevant environment. The concentration ofalbumin in human blood in vivo is approximately 0.6 mM, suggesting thatat doses of up to 500 mg, the meso-carotenoid (molecular weight 841 Da)will associate with the albumin in monomeric fashion (excludingadditional potential non-specific binding to circulating blood cells andlipoproteins, which would increase the potential non-aggregating dose).Bound meso-carotenoid molecules exhibited induced CD bands which wereadequately explained by a right-handed helical conformation of theconjugated system. Graded fluorescence quenching of HSA in the presenceof increasing concentrations of dAST reinforced the notion thatformation of carotenoid-albumin complexes were responsible for thisquenching, and suggested spatial proximity between the bound ligand andthe tryptophan 214 residue of HSA. Based on the spectroscopic data, themolecular length of the DAST molecule, and the well-characterizedcrystal structure of HSA, the binding site was tentatively assigned tothe interdomain cleft located between domains I and III.

There appears to be a positive-negative band pair in the CD spectrumabove 1:1 L/P ratio of meso-carotenoid to HSA. This finding wasattributed to intermolacular chiral exciton coupling betweenmeso-carotenoid polyene chains arranged in right-handed assembly. Theexperimental data suggested that HSA acts as a chiral template on whichthe self-assembly begins, and subsequently continues governed by thechirality of the end-groups of the meso-carotenoid molecules. Thedifferences between bisignate CD spectra obtained in pH 7.4 phosphatebuffer and Ringer solutions indicate that the self-assembly isinfluenced by the osmolarity and ionic strength of the solution. Withincreasing osmolarity, the stability of the aggregates is enhancedpresumably due to the electrostatic screening of the negatively-chargedsuccinic carboxylate functions by salt cations.

In this patent, certain U.S. patents, U.S. patent applications, andother materials (e.g., articles) have been incorporated by reference.The text of such U.S. patents, U.S. patent applications, and othermaterials is, however, only incorporated by reference to the extent thatno conflict exists between such text and the other statements anddrawings set forth herein. In the event of such conflict, then any suchconflicting text in such incorporated by reference U.S. patents, U.S.patent applications, and other materials is specifically notincorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects ofthe invention will be apparent to those skilled in the art in view ofthis description. Accordingly, this description is to be construed asillustrative only and is for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as the presently preferred embodiments. Elements andmaterials may be substituted for those illustrated and described herein,parts and processes may be reversed, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims.

REFERENCES

The following references are specifically incorporated herein byreference:

U.S. Patent Documents

-   U.S. Pat. No. 5,871,766 February 1999 Hennekens

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1. A chemical compound having the structure: a carotenoid analog orderivative having the structure

where each R³ is independently hydrogen or methyl; where each Y isindependently O or H₂; where each R is independently OR¹ or R¹; whereeach R¹ is independently -alkyl-NR² ₃ ⁺, -alkyl-NR² ₂, -aryl-NR² ₃ ⁺,-alkyl-CO₂ ⁻, -aryl-CO₂ ⁻, -alkyl-CO₂H, -alkyl-CO₂R⁶, -alkyl-CO₂R⁷,-OR⁷, -amino acid-NH₂, -amino acid-NH₃ ⁺, -phosphorylated amino acid-NH₃⁺, polyethylene glycol, dextran, H, alkyl, aryl,-alkyl-O-PO₂-O-alkyl-NR² ₃ ⁺,

where each R² is independently H, alkyl, or aryl; wherein R′ is-alkyl-O, alkyl, or aryl; wherein each R⁵ is independently H, alkyl,benzyl, or alkali salt; wherein R⁶ is alkyl,-CH₂-CH(OH)-CH₂-O-PO₂-O-alkyl-NR² ₃ ⁺,

and wherein R⁷ is -alkyl-NR² ₃ ⁺, -aryl-NR² ₃ ⁺, -alkyl-CO₂ ⁻, -aryl-CO₂⁻, -amino acid-NH₃ ⁺, -phosphorylated amino acid-NH₃ ⁺, polyethyleneglycol, dextran, H, alkyl,

or aryl.
 2. The compound of claim 1, wherein the carotenoid analog orderivative has the structure

where each X is independently -alkyl-NR⁴ ₃ ⁺, -aryl-NR⁴ ₃ ⁺, -alkyl-CO₂⁻, -aryl-CO₂ ⁻, -amino acid-NH₃ ⁺, -phosphorylated amino acid-NH₃ ⁺,polyethylene glycol, dextran, H, alkyl, aryl, or alkali salt, and whereeach R⁴ is independently H, alkyl, or aryl; where each R′ isindependently -alkyl-O, alkyl, or aryl; and where n is between 0 andabout
 12. 3. The compound of claim 1, wherein the carotenoid analog orderivative has the structure

where each X is independently -alkyl-NR⁴ ₃ ⁺, -aryl-NR⁴ ₃ ⁺, -alkyl-CO₂⁻, -aryl-CO₂ ⁻, -amino acid-NH₃ ⁺, -phosphorylated amino acid-NH₃ ⁺,polyethylene glycol, dextran, H, alkyl, aryl, or alkali salt, and whereeach R⁴ is independently H, alkyl, or aryl; where each R′ isindependently -alkyl-O, alkyl, or aryl; and where n is between 0 andabout
 12. 4. The compound of claim 1, wherein the carotenoid analog orderivative has the structure


5. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


6. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


7. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


8. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


9. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


10. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


11. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


12. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


13. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


14. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


15. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


16. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


17. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


18. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


19. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


20. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


21. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


22. The compound of claim 1, wherein each R¹ is independently


23. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


24. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


25. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure

wherein each R¹ is independently -alkyl-NR² ₃ ⁺, -alkyl-NR² ₂, -aryl-NR²₃ ⁺, -amino acid-NH₂, or -amino acid-NH₃ ⁺.
 26. The compound of claim 1,wherein the carotenoid analog or derivative has the structure

wherein each R¹ is independently -alkyl-NR² ₃ ⁺, -alkyl-NR² ₂, -aryl-NR²₃ ⁺, -amino acid-NH₂, or -amino acid-NH₃ ⁺.
 27. The compound of claim 1,wherein the carotenoid analog or derivative has the structure


28. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure


29. The compound of claim 1, wherein the carotenoid analog or derivativehas the structure